Francesca Ferraro

Mesenchymal and haematopoietic stem cells form a unique bone marrow niche

The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin+ MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent ‘mesenspheres’ that can self-renew and expand in serial transplantations. Nestin+ MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or β3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin+ cells and favours their osteoblastic differentiation, in vivo nestin+ cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin+ MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin+ cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.

Direct measurement of local oxygen concentration in the bone marrow of live animals

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.

Diabetes Impairs Hematopoietic Stem Cell Mobilization by Altering Niche Function

Impaired mobilization of hematopoietic stem cells in diabetic mice is due to sympathetic nervous system dysregulation of CXCL12 distribution. Boosting Stem Cell Mobilization Transplantation of hematopoietic stem cells (HSCs) from the bone marrow is a successful approach for treating blood diseases and certain cancers. Usually, the patient’s own (autologous) HSCs are used for transplant, but in some patients, their HSCs cannot be mobilized in sufficient numbers using the growth factor G-CSF (granulocyte colony-stimulating factor) to enable a successful transplant. In a new study, Ferraro and colleagues set out to discover the causes of this poor HSC mobilization. The investigators discovered by analyzing data from a number of bone marrow transplant patients that patients with diabetes showed poorer mobilization of HSCs in response to G-CSF than did those patients who did not have diabetes. The authors then confirmed in mouse models of type 1 and type 2 diabetes that HSCs were poorly mobilized from the bone marrow in response to G-CSF in these mice but not healthy control animals. The authors discovered that there was a defect in the bone marrow microenvironment of the diabetic mice rather than a problem with the HSCs themselves. Specifically, in diabetic (but not control) mice, the researchers observed mislocalization of HSCs in the bone marrow and an increase in the number of perivascular sympathetic nerve fibers in the niche with a concomitant inability of bone marrow mesenchymal stem cells to down-modulate production of the chemokine CXCL12 (a molecule known to mediate HSC localization). Finally, the authors were able to overcome the defect in HSC mobilization using a clinically approved drug called AMD3100 that interrupts the interaction of CXCL12 with its receptor CXCR4. The authors suggest that AMD3100 could be used to boost HSC mobilization in diabetic patients who require a bone marrow transplant. Success with transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) in patients depends on adequate collection of these cells after mobilization from the bone marrow niche by the cytokine granulocyte colony-stimulating factor (G-CSF). However, some patients fail to achieve sufficient HSPC mobilization. Retrospective analysis of bone marrow transplant patient records revealed that diabetes correlated with poor mobilization of CD34+ HSPCs. In mouse models of type 1 and type 2 diabetes (streptozotocin-induced and db/db mice, respectively), we found impaired egress of murine HSPCs from the bone marrow after G-CSF treatment. Furthermore, HSPCs were aberrantly localized in the marrow niche of the diabetic mice, and abnormalities in the number and function of sympathetic nerve termini were associated with this mislocalization. Aberrant responses to β-adrenergic stimulation of the bone marrow included an inability of marrow mesenchymal stem cells expressing the marker nestin to down-modulate the chemokine CXCL12 in response to G-CSF treatment (mesenchymal stem cells are reported to be critical for HSPC mobilization). The HSPC mobilization defect was rescued by direct pharmacological inhibition of the interaction of CXCL12 with its receptor CXCR4 using the drug AMD3100. These data suggest that there are diabetes-induced changes in bone marrow physiology and microanatomy and point to a potential intervention to overcome poor HSPC mobilization in diabetic patients.

Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo.

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

The HSC niche concept has turned 31: Has our knowledge matured?

The hematopoietic stem cell (HSC) niche is currently defined as the specific microenvironment in the bone marrow (BM) which anatomically harbors HSCs and governs their fate. It plays a pivotal role in regulating the survival and self‐renewal ability of HSCs, protecting them from exhaustion while preventing their excessive proliferation. Many different stromal cell types have been proposed as putative constituents of the niche, but their integrated function is still unrevealed. Mechanisms by which stem/progenitor cell behavior is regulated in the niche include cell‐to‐cell interaction and the production of growth factors, cytokines, and extracellular matrix proteins. The HSC niche is a dynamic entity reflecting and responding to the needs of the organism. An understanding of how the niche participates in the maintenance of tissue homeostasis and repair offers new opportunities for the development of novel therapeutic tools.

Differential Stem and Progenitor Cell Trafficking by Prostaglandin E2

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.

Concise Review: Diabetes, the Bone Marrow Niche, and Impaired Vascular Regeneration

Diabetes mellitus is a global health problem that results in multiorgan complications leading to high morbidity and mortality. Until recently, the effects of diabetes and hyperglycemia on the bone marrow microenvironment—a site where multiple organ systems converge and communicate—have been underappreciated. However, several new studies in mice, rats, and humans reveal that diabetes leads to multiple bone marrow microenvironmental defects, such as small vessel disease (microangiopathy), nerve terminal pauperization (neuropathy), and impaired stem cell mobilization (mobilopathy). The discovery that diabetes involves bone marrow‐derived progenitors implicated in maintaining cardiovascular homeostasis has been proposed as a bridging mechanism between micro‐ and macroangiopathy in distant organs. Herein, we review the physiological and molecular bone marrow abnormalities associated with diabetes and discuss how bone marrow dysfunction represents a potential root for the development of the multiorgan failure characteristic of advanced diabetes. The notion of diabetes as a bone marrow and stem cell disease opens new avenues for therapeutic interventions ultimately aimed at improving the outcome of diabetic patients.

Adult Stem Cels and Their Niches

Stem cells participate in dynamic physiologic systems that dictate the outcome of developmental events and organismal stress, Since these cells are fundamental to tissue maintenance and repair, the signals they receive play a critical role in the integrity of the organism. Much work has focused on stem cell identification and the molecular pathways involved in their regulation. Yet, we understand little about how these pathways achieve physiologically responsive stem cell functions. This chapter will review the state of our understanding of stem cells in the context of their microenvironment regarding the relation between stem cell niche dysfunction, carcinogenesis and aging.

Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells

Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted Vav1−/− HSPC showed impaired early localization near nestin+ perivascular mesenchymal stem cells; only 6.25% of Vav1−/− HSPC versus 45.8% of wild-type HSPC were located less than 30 μm from a nestin+ cell. Abnormal perivascular localization correlated with decreased retention of Vav1−/− HSPC in the bone marrow (44–60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of Vav1−/− cells vs. 53–63% for wild-type cells). The engraftment defect of Vav1−/− HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, Vav1−/− HSPC showed impaired responses to SDF1α, including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for SDF1α-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment.

Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow

Osteocalcin (Ocn)-expressing bone marrow cells produce the Notch ligand DLL4, and this is required for lymphoid progenitor cells to seed the thymus.