Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express alpha(9)beta(1) integrin. The N-terminal thrombin cleavage fragment of OPN through its binding to the alpha(9)beta(1) and alpha(4)beta(1) integrins plays a key role in the attraction, retention, regulation, and release of hemopoietic stem and progenitor cells to, in, and from their BM niche. Thrombin-cleaved OPN (trOPN) acts as a chemoattractant for stem and progenitor cells, mediating their migration in a manner that involves interaction with alpha(9)beta(1) and alpha(4)beta(1) integrins. In addition, in the absence of OPN, there is an increased number of white blood cells and, specifically, stem and progenitor cells in the peripheral circulation.

A large body of evidence suggests hemopoietic stem cells (HSCs) exist in an endosteal niche close to bone, whereas others suggest that the HSC niche is intimately associated with vasculature. In this study, we show that transplanted hemopoietic stem and progenitor cells (HSPCs) home preferentially to the trabecular-rich metaphysis of the femurs in nonablated mice at all time points from 15 minutes to 15 hours after transplantation. Within this region, they exist in an endosteal niche in close association with blood vessels. The preferential homing of HSPCs to the metaphysis occurs rapidly after transplantation, suggesting that blood vessels within this region may express a unique repertoire of endothelial adhesive molecules. One candidate is hyaluronan (HA), which is highly ex- IntroductionHemopoietic stem cells (HSCs) exist in a specialized microanatomic space within the bone marrow (BM) termed the niche. 1 Within the niche, cues from the surrounding microenvironment maintain the balance between maintenance of the stem cell pool and proliferation. Despite 30 years of research, the precise location of the HSC niche is unclear. Using different phenotypic markers for hemopoietic stem and progenitor cells (HSPCs), many groups have attempted to define the location of the HSC niche based on its cellular constituents. As examples, the endosteal or osteoblastic niche suggests that HSCs are close to or touching osteoblasts, [2][3][4][5] and the vascular niche suggests that HSCs are close to or in contact with the sinusoidal endothelium. 6,7 Considerable debate has ensued regarding the prominence of each of these niches in HSC regulation. However, several critical questions remain unanswered, including the following: Are these separate or overlapping niches? Do HSCs actually have to be in contact with particular cell types for HSC regulation? Is one cell type more important than others in regulating HSC? Of paramount importance to addressing these questions is an understanding of the three-dimensional relationship between HSCs, blood vessel (BVs), and bone.Most recently, intravital 2-photon or confocal microscopy was used to define the HSC niche by investigating the spatial distribution and lodgement of transplanted fluorescently labeled HSPCs. [7][8][9][10] However, the thick cortical bone of long bones presents a challenge for current high resolution imaging modalities as it is impervious to light. A number of innovative methodologies have recently been developed by researchers to permit imaging of HSPC homing to their niche in long bones either in vivo 8,11 or ex vivo. 10,12 These consist of cutting or splitting the bone, 10,12 grinding the cortical bone until semitransparent, 8 or inserting an imaging probe into the BM. 11 As the vasculature of the bone is continuous with the BM, 13 damaging bone disrupts BM vasculature, inducing a stress response. Stress responses include the up-regulation of the chemokine, stromal derived factor 1 (SDF-1), 14,15 and vascular cell adhesion molecule 1 (VCAM-1), 11,16 both of which a...

Hemopoietic stem cells (HSCs) reside within a specified area of the bone marrow (BM) cavity called a "niche" that modulates HSC quiescence, proliferation, differentiation, and migration.

The inherent disadvantages of using granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell (HSC) mobilization have driven efforts to identify alternate strategies based on single doses of small molecules. Here, we show targeting α9β1/α4β1 integrins with a single dose of a small molecule antagonist (BOP (N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine)) rapidly mobilizes long-term multi-lineage reconstituting HSC. Synergistic engraftment augmentation is observed when BOP is co-administered with AMD3100. Impressively, HSC in equal volumes of peripheral blood (PB) mobilized with this combination effectively out-competes PB mobilized with G-CSF. The enhanced mobilization observed using BOP and AMD3100 is recapitulated in a humanized NODSCIDIL2Rγ−/− model, demonstrated by a significant increase in PB CD34+ cells. Using a related fluorescent analogue of BOP (R-BC154), we show that this class of antagonists preferentially bind human and mouse HSC and progenitors via endogenously primed/activated α9β1/α4β1 within the endosteal niche. These results support using dual α9β1/α4β1 inhibitors as effective, rapid and transient mobilization agents with promising clinical applications.