Haematopoietic stem and progenitor cells (HSPCs) change location during development and circulate in mammals throughout life, moving into and out of the bloodstream to engage bone marrow niches in sequential steps of homing, engraftment and retention. Here we show that HSPC engraftment of bone marrow in fetal development is dependent on the guanine-nucleotide-binding protein stimulatory alpha subunit (Galpha(s)). HSPCs from adult mice deficient in Galpha(s) (Galpha(s)(-/-)) differentiate and undergo chemotaxis, but also do not home to or engraft in the bone marrow in adult mice and demonstrate a marked inability to engage the marrow microvasculature. If deleted after engraftment, Galpha(s) deficiency did not lead to lack of retention in the marrow, rather cytokine-induced mobilization into the blood was impaired. Testing whether activation of Galpha(s) affects HSPCs, pharmacological activators enhanced homing and engraftment in vivo. Galpha(s) governs specific aspects of HSPC localization under physiological conditions in vivo and may be pharmacologically targeted to improve transplantation efficiency.

To maintain lifelong production of blood cells, haematopoietic stem cells (HSCs) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSCs reside in several, perhaps overlapping, niches that produce regulatory molecules and signals necessary for homeostasis and for increased output after stress or injury. Despite considerable advances in the specific cellular or molecular mechanisms governing HSC-niche interactions, little is known about the regulatory function in the intact mammalian haematopoietic niche. Recently, we and others described a positive regulatory role for prostaglandin E2 (PGE2) on HSC function ex vivo. Here we show that inhibition of endogenous PGE2 by non-steroidal anti-inflammatory drug (NSAID) treatment in mice results in modest HSC egress from the bone marrow. Surprisingly, this was independent of the SDF-1-CXCR4 axis implicated in stem-cell migration. 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. Haematopoietic 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 other species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced E-prostanoid 4 (EP4) receptor signalling. These results not only uncover unique regulatory roles for EP4 signalling in HSC retention in the niche, but also define a rapidly translatable strategy to enhance transplantation therapeutically.

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

The avascular nature of cartilage makes it a unique tissue1-4, but whether and how the absence of nutrient supply regulates chondrogenesis remain unknown. Here we show that obstruction of vascular invasion during bone healing favours chondrogenic over osteogenic differentiation of skeletal progenitor cells. Unexpectedly, this process is driven by a decreased availability of extracellular lipids. When lipids are scarce, skeletal progenitors activate forkhead box O (FOXO) transcription factors, which bind to the Sox9 promoter and increase its expression. Besides initiating chondrogenesis, SOX9 acts as a regulator of cellular metabolism by suppressing oxidation of fatty acids, and thus adapts the cells to an avascular life. Our results define lipid scarcity as an important determinant of chondrogenic commitment, reveal a role for FOXO transcription factors during lipid starvation, and identify SOX9 as a critical metabolic mediator. These data highlight the importance of the nutritional microenvironment in the specification of skeletal cell fate.

Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis, expression of hypoxia inducible factor-1α (Hif-1α) and related genes, and staining with surrogate hypoxic markers (for example, pimonidazole). Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.