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.

Characterizing how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for therapeutic manipulation of stem cells1. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types2–4. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis5, expression of HIF-1 and related genes6, and staining with surrogate hypoxic markers (e.g. pimonidazole)6–8. Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow (BM) of live mice. Using two-photon phosphorescence lifetime microscopy (2PLM), we determined the absolute pO2 of the BM to be quite low (<32 mmHg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (~9.9 mmHg, 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 dramatically after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Autologous hematopoietic stem/progenitor cells (HSPC) transplantation success depends upon adequate cell collection after G-CSF-administration that a substantial fraction of patients fails to achieve. Retrospective analysis of patient records demonstrated that diabetes correlated with lower CD34+ cell mobilization. Using mouse models, we found impaired HSPC egress from the bone marrow in either streptozotocin-induced or db/db diabetic animals. HSPC aberrantly localized within the marrow microenvironment of diabetic animals in association with abnormalities in sympathetic neuron number and function. Markedly increased sympathetic neuron density was accompanied by abnormal response to β-adrenergic stimulation and a failure to generate the G-CSF-induced CXCL12 gradient in nestin-expressing mesenchymal cells associated with HSPC mobilization. Alternative mobilization by direct pharmacologic inhibition of CXCL12-CXCR4 interaction rescued the defect. These data reveal diabetes-induced changes in bone marrow physiology and microanatomy and point to a pathophysiologically based approach to overcome HSPC mobilization defects in diabetic patients.

SUMMARY AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ∼40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.

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

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 supported by the bone marrow hematopoietic microenvironment to maintain long-term self-renewal. [1][2][3] Leukemia stem cells (LSCs) possess the ability to initiate, maintain, and serially propagate leukemia in vivo. Furthermore, LSCs infiltrate the bone marrow 4,5 and interfere with the normal HSC-microenvironment homeostasis. 6 Available data indicate that LSCs also interact with the hematopoietic microenvironment to maintain self-renewal and to mitigate the effects of cytotoxic chemotherapy. 5,[7][8][9] Thus, disruption of LSC-niche interactions may have therapeutic value, as observed by an enhanced sensitivity to cytotoxic chemotherapy after leukemia mobilization. [8][9][10] Leukemia is the consequence of stepwise genetic alterations that confer both proliferative and survival advantage, as well as self-renewal to the malignant cells. 11 A recognized early stage of LSC development is the pre-LSC stage composed of immortalized hematopoietic stem and progenitor cells that give rise to leukemia in vivo with variable latency, presumably because of the gradual accumulation of additional genetic hits. 12,13 In contrast, LSCs (derived from mice with established leukemia) give rise to fully penetrant, short-onset leukemia in secondary recipients. 14 Biologically, pre-LSCs and LSCs are distinctive in their relative pace of disease onset and leukemogenic potential. However, it is not known whether this distinction corresponds to disparate requirements for cell-extrinsic signaling from the bone marrow microenvironment or whether a potential pre-LSC or LSC niche would overlap with that of normal HSCs.A spectrum of signaling pathways have been demonstrated to regulate the interactions of HSCs with the bone marrow microenvironment. 7,15 However, the cellular and molecular components of the LSC microenvironment remain poorly understood. Dysregulation of the canonical Wnt signaling pathway is known to constrain HSC function in vivo. 16,17 Furthermore, canonical Wnt signaling is activated in some acute myeloid leukemia (AML) LSCs, and targeted genetic deletion of the downstream Wnt effector ␤-catenin inhibits leukemogenesis. 12,13 Moreover, cellextrinsic inhibition of Wnt signaling through ectopic DKK1 expression impairs leukemia cell proliferation in vitro. 18 We used a syngeneic murine model of MLL-AF9-induced AML to determine the localization of pre-LSCs and LSCs within the bone marrow hematopoietic microenvironment at different stages of leukemic progression and analyzed the effect of cellintrinsic and cell-extrinsic alterations of Wnt signaling on pre-LSC and LSC niche requirements. For personal use only. on May 10, 2018. by guest www.bloodjournal.org From Methods Flow cytometryCells were stained with a lineage cocktail of biotin-labeled antimouse antibodies (Ter119, CD3, CD4, CD8, B220, Mac1, and Gr1; BD Biosciences PharMingen). Lineage-positive cells were depleted with Dynabeads (Invitrogen). Lineage-depleted cells were stained with c-kit (2B8), Sca-1 (D7), CD34 (RAM34),...

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.