The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). Here, we demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP) of HSCs, which adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 with its ligand Angiopoietin-1 (Ang-1) induced cobblestone formation of HSCs in vitro and maintained in vivo long-term repopulating activity of HSCs. Furthermore, Ang-1 enhanced the ability of HSCs to become quiescent and induced adhesion to bone, resulting in protection of the HSC compartment from myelosuppressive stress. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche.

Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is crucial for the maintenance of hematopoietic homeostasis. Here we show that activation of p38 MAPK in response to increasing levels of reactive oxygen species (ROS) limits the lifespan of HSCs in vivo. In Atm(-/-) mice, elevation of ROS levels induces HSC-specific phosphorylation of p38 MAPK accompanied by a defect in the maintenance of HSC quiescence. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity and in the maintenance of HSC quiescence, indicating that the ROS-p38 MAPK pathway contributes to exhaustion of the stem cell population. Furthermore, prolonged treatment with an antioxidant or an inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial transplantation experiments. These data show that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Our characterization of molecular mechanisms that limit HSC lifespan may lead to beneficial therapies for human disease.

The 'ataxia telangiectasia mutated' (Atm) gene maintains genomic stability by activating a key cell-cycle checkpoint in response to DNA damage, telomeric instability or oxidative stress. Mutational inactivation of the gene causes an autosomal recessive disorder, ataxia-telangiectasia, characterized by immunodeficiency, progressive cerebellar ataxia, oculocutaneous telangiectasia, defective spermatogenesis, premature ageing and a high incidence of lymphoma. Here we show that ATM has an essential function in the reconstitutive capacity of haematopoietic stem cells (HSCs) but is not as important for the proliferation or differentiation of progenitors, in a telomere-independent manner. Atm-/- mice older than 24 weeks showed progressive bone marrow failure resulting from a defect in HSC function that was associated with elevated reactive oxygen species. Treatment with anti-oxidative agents restored the reconstitutive capacity of Atm-/- HSCs, resulting in the prevention of bone marrow failure. Activation of the p16(INK4a)-retinoblastoma (Rb) gene product pathway in response to elevated reactive oxygen species led to the failure of Atm-/- HSCs. These results show that the self-renewal capacity of HSCs depends on ATM-mediated inhibition of oxidative stress.

Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Mammalian HSCs are kept quiescent in the endosteal niche, a hypoxic zone of the bone marrow (BM). In this study, we show that normal HSCs maintain intracellular hypoxia and stabilize hypoxia-inducible factor-1alpha (HIF-1alpha) protein. In HIF-1alpha-deficient mice, the HSCs lost their cell cycle quiescence and HSC numbers decreased during various stress settings including bone marrow transplantation, myelosuppression, or aging, in a p16(Ink4a)/p19(Arf)-dependent manner. Overstabilization of HIF-1alpha by biallelic loss of an E3 ubiquitin ligase for HIF-1alpha (VHL) induced cell cycle quiescence in HSCs and their progenitors but resulted in an impairment in transplantation capacity. In contrast, monoallelic loss of VHL induced cell cycle quiescence and improved BM engraftment during bone marrow transplantation. These data indicate that HSCs maintain cell cycle quiescence through the precise regulation of HIF-1alpha levels.

Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone marrow niche. Here we show that Foxo3a, a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway, is critical for HSC self-renewal. We generated gene-targeted Foxo3a(-/-) mice and showed that, although the proliferation and differentiation of Foxo3a(-/-) hematopoietic progenitors were normal, the number of colony-forming cells present in long-term cocultures of Foxo3a(-/-) bone marrow cells and stromal cells was reduced. The ability of Foxo3a(-/-) HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired. Foxo3a(-/-) HSCs also showed increased phosphorylation of p38MAPK, an elevation of ROS, defective maintenance of quiescence, and heightened sensitivity to cell-cycle-specific myelotoxic injury. Finally, HSC frequencies were significantly decreased in aged Foxo3a(-/-) mice compared to the littermate controls. Our results demonstrate that Foxo3a plays a pivotal role in maintaining the HSC pool.

Defining the metabolic programs that underlie stem cell maintenance will be essential for developing strategies to manipulate stem cell capacity. Mammalian hematopoietic stem cells (HSCs) maintain cell cycle quiescence in a hypoxic microenvironment. It has been proposed that HSCs exhibit a distinct metabolic phenotype under these conditions. Here we directly investigated this idea using metabolomic analysis and found that HSCs generate adenosine-5'-triphosphate by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Elevated Pdk expression leads to active suppression of the influx of glycolytic metabolites into mitochondria. Pdk overexpression in glycolysis-defective HSCs restored glycolysis, cell cycle quiescence, and stem cell capacity, while loss of both Pdk2 and Pdk4 attenuated HSC quiescence, glycolysis, and transplantation capacity. Moreover, treatment of HSCs with a Pdk mimetic promoted their survival and transplantation capacity. Thus, glycolytic metabolic status governed by Pdk acts as a cell cycle checkpoint that modulates HSC quiescence and function.

Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) reside in a hypoxic bone marrow environment, and their metabolic status is distinct from that of their differentiated progeny. HSCs generate energy mainly via anaerobic metabolism by maintaining a high rate of glycolysis. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species, but leaves HSCs susceptible to changes in redox status. In this review, we discuss the importance of oxygen homeostasis and energy metabolism for maintenance of HSC function and long-term self-renewal.

Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.