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Hematopoiesis in mammals undergoes a developmental shift in location from fetal liver to bone marrow accompanied by a gradual transition from highly proliferative to deeply quiescent stem cell populations. P2Y receptors are G-protein-coupled nucleotide receptors participating in vascular and immune responses to injury. We identified a P2Y-like receptor for UDP-conjugated sugars, GPR105 (P2Y 14 ), with restricted expression on primitive cells in the hematopoietic lineage. Anti-GPR105 antibody selectively isolated a subset of hematopoietic cells within the fetal bone marrow, but not in the fetal liver, that was enriched for G0 cell cycle status and for in vitro stem-cell-like multipotential long-term culture capability. Conditioned media from bone marrow stroma induced receptor activation and chemotaxis that was sensitive to G␣i and anti-receptor antibody inhibition. GPR105 is a G-protein-coupled receptor identifying a quiescent, primitive population of hematopoietic cells restricted to bone marrow. It mediates primitive cell responses to specific hematopoietic microenvironments and extends the known immune system functions of P2Y receptors to the stem cell level. These data suggest a new class of receptors participating in the regulation of the stem cell compartment. G-protein-coupled receptors (GPRs) have a broad repertoire of activating ligands ranging from photons to chemokines and induce an array of cellular events in virtually every physiologic system. Yet there remains a large proportion of GPR without known ligands or with limited known functions. A group of GPRs responsive to nucleotides (termed P2Y receptors) has been defined mediating cell-cell communication in the nervous system and in modulating vascular tone . A well-defined effect of nucleotides on platelet activation and vascular smooth muscle migration and growth has suggested participation of P2Y receptors in the response to injury. More recently, these receptors have been noted to affect cellular constituents of the innate immune system altering functional characteristics of monocytes, eosinophils, and dendritic cells and to play a critical role in terminating the inflammatory response in vivo (Mutini et al. 1999;Ferrari et al. 2000;Idzko et al. 2001;Santiago-Perez et al. 2001;Warny et al. 2001;Wilkin et al. 2001). The P2Y receptor specificity originally thought to be restricted to purine (adenine) nucleotides has been extended to pyrimidine nucleotides (uridine) and more recently to a receptor with specificity for UDP, but only when conjugated to glucose or related sugars (Chambers et al. 2000). This receptor, GPR105 (recently designated P2Y 14 ; Abbracchio et al. 2003), was originally noted to be expressed in rat brain tegmentum, but has no known function apart from being the presumed basis for UDP-glucose to induce diaphragmatic contraction or neural action potentials (Pastoris et al. 1979(Pastoris et al. , 1981. We provide evidence that this receptor participates in regulation of hematopoietic cells with stem cell characteristics.

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EphB4 (HTK) and its ligand, ephrinB2, are critical for angiogenesis and result in fatal abnormalities of capillary formation in null mice. EphB4 was originally identified in human bone marrow CD34 ؉ cells by us and has since been reported to be expressed in erythroid progenitors, whereas the ligand ephrinB2 is expressed in bone marrow stromal cells. Reasoning that the developmental relationship between angiogenesis and hematopoiesis implies common regulatory molecules, we assessed whether EphB4 signaling influences the function and phenotype of primitive human hematopoietic cells. IntroductionHematopoietic stem cells (HSCs) have the capacity to self-renew and to differentiate along a number of pathways, thereby generating all blood cells. Understanding the molecular mechanisms that regulate the formation, growth, and differentiation of HSCs has become increasingly complex. 1 The bone marrow (BM) is a diverse environment that contains a variety of different cell types and extracellular matrix molecules. The BM microenvironment produces a wide range of stimuli to different hematopoietic cells, providing combinatorial relationships that result in the finely tuned hematopoietic system. How factors (in either membrane-bound or soluble form) produced by BM stromal cells regulate the balance of self-renewal and differentiation of specific blood cell lineages remains a major question.Eph receptor tyrosine kinases and their ligands, ephrins, play important roles in various processes during embryonic development, including the targeting behavior of migratory neurons, vascular cell assembly, and angiogenesis. 2 Fourteen Eph receptors have been catalogued into EphA or EphB subclasses based on their affinity for ligands. Eight ephrins have been identified to date. They are membrane proteins of either glycerophosphatidylinsitol (GPI)-linked (ephrinA) or transmembrane (ephrinB). 3 Rather than longrange communication, signaling from Eph receptors and their ligands is restricted to sites of direct cell-cell contact and is capable of inducing reciprocal bidirectional events between interacting cells. 4 Some Eph and ephrin molecules have been found to be expressed in the hematopoietic system. EphA3 (Hek) was originally cloned from a pre-B-cell leukemia and is expressed in some T-cell lines. 5 EphA1 (Esk), EphA2 (Eck), and EphB2 (Hek5) were reported to be expressed in thymus. 6-8 EphA4 (Hek8) and EphA7 (Hek11) appear to be expressed in human fetal bone marrow pro-B cells. 9 The receptor tyrosine kinase EphB4 (HTK) and its cognate ligand, ephrinB2 (HTKL) are widely expressed in fetal and adult tissues. 10,11 Unlike most of the Eph subfamily members, EphB4 does not appear to be expressed in the central nervous system. Recent studies have shown that EphB4 is specifically expressed at the venous endothelium, whereas ephrinB2 is specifically and reciprocally expressed on arterial endothelial cells at the earliest stages of vascular development. 12 Mice lacking either EphB4 or ephrinB2 display identical defects in angiogenesis by arteri...