Significance: Blood forming, hematopoietic stem cells (HSCs) mostly reside in the bone marrow in a quiescent, nonmotile state via adhesion interactions with stromal cells and macrophages. Quiescent, proliferating, and differentiating stem cells have different metabolism, and accordingly different amounts of intracellular reactive oxygen species (ROS). Importantly, ROS is not just a byproduct of metabolism, but also plays a role in stem cell state and function. Recent Advances: ROS levels are dynamic and reversibly dictate enhanced cycling and myeloid bias in ROS high short-term repopulating stem cells, and ROS low quiescent long-term repopulating stem cells. Low levels of ROS, regulated by intrinsic factors such as cell respiration or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) activity, or extrinsic factors such as stem cell factor or prostaglandin E2 are required for maintaining stem cell self-renewal. High ROS levels, due to stress and inflammation, induce stem cell differentiation and enhanced motility. Critical Issues: Stem cells need to be protected from high ROS levels to avoid stem cell exhaustion, insufficient host immunity, and leukemic transformation that may occur during chronic inflammation. However, continuous low ROS production will lead to lack of stem cell function and opportunistic infections. Ultimately, balanced ROS levels are crucial for maintaining the small stem cell pool and host immunity, both in homeostasis and during stress situations. Future Directions: Deciphering the signaling pathway of ROS in HSC will provide a better understanding of ROS roles in switching HSC from quiescence to activation and vice versa, and will also shed light on the possible roles of ROS in leukemia initiation and development. Antioxid. Redox Signal. 21, 1605-1619.

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1 ؉ /c-Kit ؉ /Lin ؊ (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P 1 , or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit ؉ /Lin ؊ cells expressing surface S1P 1 in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin ؉ mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P 1 axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility. (Blood. 2012;119(11):2478-2488) IntroductionMotility is a key feature of hematopoietic stem and progenitor cells (HSPCs). These cells are continuously released at basal levels from the bone marrow (BM) reservoir to the circulation during steady state homeostasis together with maturing leukocytes, and at increased rates on stress situations, such as bleeding or inflammation. 1,2 The complex process of HSPC trafficking is orchestrated by various cytokines, chemokines, proteolytic enzymes, and adhesion molecules 3-5 through a dynamic interplay between the immune and nervous systems within the bone microenvironment. 1,2,6-8 HSPC mobilization can be clinically induced by a variety of cytokines and drugs, such as granulocyte colony stimulating factor (G-CSF, the most commonly used agent), 9,10 sulfated polysaccharides, 11,12 and recently also by AMD3100. 13,14 Repetitive G-CSF administrations cause mobilization by inducing proliferation and differentiation of HSPC, thus increasing their pool size, accompanied by reduced retention in the BM microenvironment. 15 The chemokine stromal cell-derived factor-1 (SDF-1, also termed CXCL12), which is the most powerful chemoattractant of both human and murine HSPCs, 16,17 and its major receptor CXCR4 are key players in HSPC mobilization. 10,12,13,[18][19][20][21] SDF-1 is transiently increased in the murine BM during G-CSF stimulation followed by its downregulation at both protein 18,22 and mRNA 23 levels, reaching a nadir at the peak of HSPC mobilization. 18 The intensified SDF-1/CXCR4 interactions induce enhanced production of reactive oxygen species (ROS) through activation of the HGF/c-Met path...

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation-related also control retention of EPCR+ LT-HSCs in the bone marrow and their recruitment to the blood via two different protease activated receptor 1 (PAR1)-mediated pathways. Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to TACE-mediated EPCR shedding, enhanced CXCL12-CXCR4-induced motility, and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with protein C that retain EPCR+ LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing VLA4 affinity and adhesion. Inhibition of NO production by activated protein C (aPC)-EPCR-PAR1 signaling reduces progenitor cell egress, increases NOlow bone marrow EPCR+ LT-HSCs retention and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR that control NO production to balance maintenance and recruitment of bone marrow EPCR+ LT-HSCs with clinical relevance.

The metabolic state of stem cells is emerging as an important determinant of their fate. In the bone marrow, haematopoietic stem cell (HSC) entry into cycle, triggered by an increase in intracellular reactive oxygen species (ROS), corresponds to a critical metabolic switch from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Here we show that loss of mitochondrial carrier homologue 2 (MTCH2) increases mitochondrial OXPHOS, triggering HSC and progenitor entry into cycle. Elevated OXPHOS is accompanied by an increase in mitochondrial size, increase in ATP and ROS levels, and protection from irradiation-induced apoptosis. In contrast, a phosphorylation-deficient mutant of BID, MTCH2's ligand, induces a similar increase in OXPHOS, but with higher ROS and reduced ATP levels, and is associated with hypersensitivity to irradiation. Thus, our results demonstrate that MTCH2 is a negative regulator of mitochondrial OXPHOS downstream of BID, indispensible in maintaining HSC homeostasis.

Hematopoietic stem and progenitor cells (HSPCs) are regulated by various bone marrow stromal cell types. Here we identified rare activated bone marrow monocytes and macrophages with high expression of α-smooth muscle actin (α-SMA) and the cyclooxygenase COX-2 that were adjacent to primitive HSPCs. These myeloid cells resisted radiation-induced cell death and further upregulated COX-2 expression under stress conditions. COX-2-derived prostaglandin E(2) (PGE(2)) prevented HSPC exhaustion by limiting the production of reactive oxygen species (ROS) via inhibition of the kinase Akt and higher stromal-cell expression of the chemokine CXCL12, which is essential for stem-cell quiescence. Our study identifies a previously unknown subset of α-SMA(+) activated monocytes and macrophages that maintain HSPCs and protect them from exhaustion during alarm situations.

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and IntroductionDuring steady state conditions, adhesive interactions between the bone marrow (BM) stromal cells and primitive hematopoietic cells mostly result in stem cell retention, in a noncycling and nonmotile mode. However, low levels of progenitor cells are continuously released from the BM to the blood circulation as part of homeostasis. This process is dramatically amplified during emergency situations because of damage and cell death, as part of host defense and repair, in response to stress signals, including cytokines such as granulocyte colony-stimulating factor (G-CSF). Repetitive G-CSF stimulations are commonly used in the clinic, mimicking emergency situations to harvest stem and progenitor cells from the circulation for transplantation protocols. 1,2 The BM reservoir of immature and maturing leukocytes is dynamic, replenishing the blood with new cells on demand. These dynamic changes are achieved through a complex interplay between the immune and nervous systems, the bones and the BM microenvironment, involving cytokines, chemokines, proteolytic enzymes, and adhesion molecules. 3 In particular, oscillations in BM levels of stromal cell-derived factor 1 (SDF-1; transiently increased and subsequently degraded) and CXC chemokine receptor-4 (CXCR4) activation play a crucial role in promoting progenitor cell egress. 4,5 The cytokine hepatocyte growth factor (HGF) and its receptor c-Met control complex biologic programs known as "invasive growth" and tumor spreading. 6 Reactive oxygen species (ROS) are constantly generated during intracellular metabolism and in response to cytokines. Although excess ROS can cause oxidative damage to DNA, moderate levels have important roles in cell signaling, regulating different physiologic and pathologic cellular processes, including cell-cycle progression, migration, and invasion. 7 Finally, redox signaling has emerged as an important regulator of hematopoietic stem cell (HSC) self-renewal and lifespan. 8,9 The Forkhead Box, class O (FOXO) family of Forkhead transcription factors is a regulator of oxidative stress. 10 Loss of FOXO function in HSCs results in increased ROS levels, defective maintenance of quiescence, and reduced long-term repopulating ability. 11,12 FOXOs are a direct substrate of the protein kinase Akt, a mammalian target of rapamycin inhibition (mTOR) target, 13 which inactivates them by phosphorylation. 14 In this study, we demonstrate that c-Met expression levels on immature and maturing leukocytes in the BM reservoir are dynamic and dramatically increased when urgent requirements for enhanced leukocyte production and recruitment emerge. Moreover, full c-Me...

The chemokine CXCL12 is essential for the function of hematopoietic stem and progenitor cells. Here we report that secretion of functional CXCL12 from human bone marrow stromal cells (BMSCs) was a cell contact-dependent event mediated by connexin-43 (Cx43) and Cx45 gap junctions. Inhibition of connexin gap junctions impaired the secretion of CXCL12 and homing of leukocytes to mouse bone marrow. Purified human CD34(+) progenitor cells did not adhere to noncontacting BMSCs, which led to a much smaller pool of immature cells. Calcium conduction activated signaling by cAMP-protein kinase A (PKA) and induced CXCL12 secretion mediated by the GTPase RalA. Cx43 and Cx45 additionally controlled Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1. We suggest that BMSCs form a dynamic syncytium via connexin gap junctions that regulates CXC12 secretion and the homeostasis of hematopoietic stem cells.

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