The microenvironment, or niche, surrounding a stem cell largely governs its cellular fate. Two anatomical niches for hematopoietic stem cells (HSCs) have been reported in the bone marrow, but a distinct function for each of these niches remains unclear. Here we report a new role for the adhesion molecule E-selectin expressed exclusively by bone marrow endothelial cells in the vascular HSC niche. HSC quiescence was enhanced and self-renewal potential was increased in E-selectin knockout (Sele(-/-)) mice or after administration of an E-selectin antagonist, demonstrating that E-selectin promotes HSC proliferation and is a crucial component of the vascular niche. These effects are not mediated by canonical E-selectin ligands. Deletion or blockade of E-selectin enhances HSC survival threefold to sixfold after treatment of mice with chemotherapeutic agents or irradiation and accelerates blood neutrophil recovery. As bone marrow suppression is a severe side effect of high-dose chemotherapy, transient blockade of E-selectin is potentially a promising treatment for the protection of HSCs during chemotherapy or irradiation.

Human embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which reside in a 3–5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen-dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced expression of SOX2, NANOG and POU5F1 (OCT4) mRNA as well as POU5F1 protein compared with hypoxic conditions. HIF1A protein was not expressed at 20% oxygen and displayed only a transient, nuclear localisation at 5% oxygen. HIF2A (EPAS1) and HIF3A displayed a cytoplasmic localisation during initial hypoxic culture but translocated to the nucleus following long-term culture at 5% oxygen and were significantly upregulated compared with cells cultured at 20% oxygen. Silencing of HIF2A resulted in a significant decrease in both hES cell proliferation and POU5F1, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1, was concomitantly increased. HIF3A upregulated HIF2A and prevented HIF1A expression with the knockdown of HIF3A resulting in the reappearance of HIF1A protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a highly proliferative, pluripotent population of hES cells. While HIF3A was found to regulate the expression of both HIF1A and HIF2A, it is HIF2A which regulates hES cell pluripotency as well as proliferation under hypoxic conditions.

The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide (CYP) as adjuvant to granulocyte colonystimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. It has recently emerged that G-CSF mediates HSC mobilization and inhibits bone formation via specific bone marrow (BM) macrophages. We compared the effect of these three mobilizing agents on BM macrophages, bone formation, osteoblasts, HSC niches and HSC reconstitution potential. Both G-CSF and CYP suppressed niche-supportive macrophages and osteoblasts, and inhibited expression of endosteal cytokines resulting in major impairment of HSC reconstitution potential remaining in the mobilized BM. In sharp contrast, although AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, endosteal cytokine expression or reconstitution potential of HSC remaining in the mobilized BM.In conclusion, although G-CSF, CYP and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches and bone formation are distinct with both G-CSF and CYP targeting HSC niche function and bone formation, whereas AMD3100 directly targets HSC without altering niche function or bone formation.

ABSTRACTinflammation on bone, hematopoietic and erythropoietic maintenance and agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

Key Points• HIF-1␣ protein stabilization increases HSC quiescence in vivo.• HIF-1␣ protein stabilization increases HSC resistance to irradiation and accelerates recovery. IntroductionTo remain in an undifferentiated state, hematopoietic stem cells (HSCs) need be lodged in specific niches of the BM where they can preserve their essential capacity to self-renew and reconstitute the whole hematopoietic and immune systems on transplantation. 1-2 In mice 3 and humans, 4 the BM contains 2 pools of HSCs: (1) a quiescent pool that divides very infrequently approximately every 145 days to self-renew and maintain a genetic reserve and (2) an activated pool that divides more frequently for the daily replacement of hematopoietic progenitor cells (HPCs), blood leukocytes, erythrocytes, and platelets. Molecular components of HSC niches are critical to maintaining the correct balance among quiescence, self-renewing proliferation, and differentiation of HSCs. It has been found recently that, in addition to the stromal cells forming niches and the arrays of essential mediators they secrete, the physicochemical conditions within niches are critical to maintaining HSC quiescence and self-renewal. 5 For example, the most quiescent HSCs capable of serial reconstitution in serial transplantations reside in niches very poorly perfused by the circulating blood, whereas more active and proliferative HSCs capable of only a single round of transplantation or reconstitution reside in more perfused niches. 6 A direct consequence of low perfusion could be increased local hypoxia. Indeed, the oxygenation rate of a tissue is dependent on how rapidly oxygen solubilized in the circulating blood perfuses into the tissue and how rapidly this oxygen is consumed by cells in an active metabolic state. [7][8] Similar to the BM, solid tumors are sites of rapid regeneration and cell division. Analyses of blood perfusion and hypoxia in solid tumors have shown that areas that are poorly perfused are stained by the hypoxia sensitive marker pimonidazole, suggesting a hypoxic state, 9 and contain tumor stem cells. [10][11] Similarly, the endosteal region of the mouse BM, which is known to harbor niches containing quiescent HSCs,3,[12][13][14][15] is also stained by pimonidazole in steady-state conditions, also suggesting a hypoxic state. [16][17] A functional consequence of tissue hypoxia is the stabilization of a family of oxygen-labile transcription factors called hypoxiainducing factors (HIFs). HIFs are heterodimers of an O 2 -labile ␣-subunit, and a stable ␤-subunit called the aryl hydrocarbon receptor nuclear translocator (ARNT). Once the HIF-␣:ARNT complex is formed, it can then translocate to the nucleus and activate the transcription of genes containing hypoxia-responsive elements. Hematopoietic cells, including HSCs, express HIF-1␣ mRNA. 18 In hypoxic conditions with an oxygen concentration below 2%, the HIF-␣ protein is stable and the complex with ARNT is formed, translocates to the nucleus, and initiates transcription of hypoxia-responsive elemen...

Energy metabolism is intrinsic to cell viability but surprisingly has been little studied in human embryonic stem cells (hESCs). The current study aims to investigate the effect of environmental O2 tension on carbohydrate utilisation of hESCs. Highly pluripotent hESCs cultured at 5% O2 consumed significantly more glucose, less pyruvate and produced more lactate compared to those maintained at 20% O2. Moreover, hESCs cultured at atmospheric O2 levels expressed significantly less OCT4, SOX2 and NANOG than those maintained at 5% O2. To determine whether this difference in metabolism was a reflection of the pluripotent state, hESCs were cultured at 5% O2 in the absence of FGF2 for 16 hours leading to a significant reduction in the expression of SOX2. In addition, these cells consumed less glucose and produced significantly less lactate compared to those cultured in the presence of FGF2. hESCs maintained at 5% O2 were found to consume significantly less O2 than those cultured in the absence of FGF2, or at 20% O2. GLUT1 expression correlated with glucose consumption and using siRNA and chromatin immunoprecipitation was found to be directly regulated by hypoxia inducible factor (HIF)-2α at 5% O2. In conclusion, highly pluripotent cells associated with hypoxic culture consume low levels of O2, high levels of glucose and produce large amounts of lactate, while at atmospheric conditions glucose consumption and lactate production are reduced and there is an increase in oxidative metabolism. These data suggest that environmental O2 regulates energy metabolism and is intrinsic to the self-renewal of hESCs.

Hypoxia-inducible factor (HIF)-1α accumulation promotes hematopoietic stem cells' quiescence and is necessary to maintain their self-renewal. However, the role of HIF-2α in hematopoietic cells is less clear. We investigated the role of HIF-2α in leukemia and lymphoma cells. HIF-2α expression was high in subsets of human and mouse leukemia and lymphoma cells, whereas it was low in normal bone marrow leukocytes. To investigate the role of HIF-2α, we transduced human HIF-2α cDNA in mouse syngeneic models of myeloid preleukemia and a transgenic model of B lymphoma. Ectopic expression of HIF-2α accelerated leukemia cell proliferation in vitro. Mice transplanted with cells transduced with HIF-2α died significantly faster of leukemia or B lymphoma than control mice transplanted with empty vector-transduced cells. Conversely, HIF-2α knockdown in human myeloid leukemia HL60 cells decreased proliferation in vitro and significantly prolonged animal survival following transplantation. In human acute myeloid leukemia (AML), HIF-2α mRNA was significantly elevated in several subsets such as the t(15;17), inv(16), complex karyotype and favorable cytogenetic groups. However, patients with high HIF-2α expression had a trend to higher disease-free survival in univariate analysis. The different effects of HIF-2α overexpression in mouse models of leukemia and human AML illustrates the complexity of this mutliclonal disease.

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