SUMMARY To maintain lifelong production of blood cells, hematopoietic stem cells (HSC) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSC (LT-HSC) reside in several, perhaps overlapping, niches that produce regulatory molecules/signals necessary for homeostasis and increased output following stress/injury 1–5. Despite significant advances in specific cellular or molecular mechanisms governing HSC/niche interactions, little is understood about regulatory function within the intact mammalian hematopoietic niche. Recently, we and others described a positive regulatory role for Prostaglandin E2 (PGE2) on HSC function ex vivo 6,7. While exploring the role of endogenous PGE2 we unexpectedly observed hematopoietic egress after nonsteroidal anti-inflammatory drug (NSAID) treatment. Surprisingly, this was independent of the SDF-1/CXCR4 axis. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin (OPN). Hematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in higher species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced EP4 receptor signaling. These results not only uncover unique regulatory roles for EP4 signaling in HSC retention in the niche but also define a rapidly translatable strategy to therapeutically enhance transplantation.

IntroductionHematopoietic stem cells (HSCs) are multipotent progenitor cells that give rise to all types of mature blood cells. HSCs reside in a complex cellular microenvironment containing osteoblasts (OBs), osteoclasts, endothelial cells, stromal cells (SCs), mesenchymal progenitor cells, and adipocytes as well as multiple components of the extracellular matrix. Collectively, these cellular elements and the extracellular matrix constitute the hematopoietic niche, which most probably regulates the size of the stem cell pool and controls HSC fate. 1 OBs play a critical role in HSC function and self-renewal. Primitive HSCs that are in association with the endosteal region have high proliferative and repopulating capacities. 2 OBs can deliver proliferative signals to HSCs during mobilization. 3 Human OBs secrete cytokines, such as granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and leukemia inhibitory factor, thereby supporting hematopoietic progenitor cell (HPC) function in vitro. [4][5][6] Furthermore, OBs secrete angiopoietin, thrombopoietin, and stromal cell-derived factor-1, all of which regulate HSC maintenance. [7][8][9] Physical and molecular interactions between HPCs and OBs supported in vitro hematopoiesis 5 and survival, 10 whereas cotransplantation of OBs with HSCs improved engraftment. 11 However, others questioned whether OBs contribute to the formation of niches where vascular and perivascular cells play a major role in maintaining HSC function. 12 In addition to stem cell-enhancing activity, microenvironment cells in multiple systems can down-regulate stem cell function.Endothelial cells in the perivascular niche reduce the adipogenic potential of adipose stromal cells by up-regulating inhibitors of adipogenesis. 13 In the hematopoietic system, adipocytes inhibit lineage-specific differentiation 14 and engraftment of transplanted cells. 15 These observations suggest that different cells of the hematopoietic niche mediate both positive and negative effects on stem and progenitor cells.Notch signaling is crucial for HSC formation during embryonic development 16 and is critical for HSC maintenance. 17 Notch signaling regulates differentiation and maintenance of HSCs, and Notch1 activation promotes stem cell self-renewal. 18 Calvi 19 and Weber et al 20 demonstrated the role of the endosteal niche in maintaining HSC self-renewal through the activation of Notch receptors on HSCs by Jagged1 expressed by OBs. However, the role of Notch signaling in HSC homeostasis has been questioned 21,22 because impeding key signaling molecules was ineffective in immediately decreasing HSC numbers or suppressing hematopoiesis.At present, we do not know precisely how different cellular elements of the hematopoietic niche collaborate to promote HSC self-renewal and to maintain the stem cell pool. Similarly, the interplay between different cell types of the hematopoietic niche that promotes or impedes self-renewing signaling pathways is also not well understood. Herein, we investigat...

Key Points• CD166 identifies human and murine long-term repopulating stem cells.• CD166 is a functional marker of stem cells and the hematopoietic niche.We previously showed that immature CD166

Multiple myeloma (MM) is incurable once osteolytic lesions have seeded at skeletal sites, but factors mediating this deadly pathogenic advance remain poorly understood. Here we report evidence of a major role for the cell adhesion molecule CD166, which we discovered to be highly expressed in MM cell lines and primary bone marrow (BM) cells from patients. CD166+ MM cells homed more efficiently than CD166− cells to the BM of engrafted immunodeficient NSG mice. CD166 silencing in MM cells enabled longer survival, a smaller tumor burden and less osteolytic lesions, as compared to mice bearing control cells. CD166 deficiency in MM cell lines or CD138+ BM cells from MM patients compromised their ability to induce bone resorption in an ex vivo organ culture system. Further, CD166 deficiency in MM cells also reduced formation of osteolytic disease in vivo after intra-tibial engraftment. Mechanistic investigation revealed that CD166 expression in MM cells inhibited osteoblastogenesis of BM-derived osteoblast progenitors by suppressing RUNX2 gene expression. Conversely, CD166 expression in MM cells promoted osteoclastogenesis by activating TRAF6-dependent signaling pathways in osteoclast progenitors. Overall, our results define CD166 as a pivotal director in MM cell homing to the BM and MM progression, rationalizing its further study as a candidate therapeutic target for MM treatment.

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