We report transplanted hemopoietic stem cells (HSC) preferentially lodge within two cells of mature megakaryocytes (MM). With both populations comprising ~0.2% of bone marrow cells, this strongly suggests a key functional interaction. HSC isolated from the endosteum (eLSKSLAM) showed significantly increased hemopoietic cell proliferation while in co-culture with MM. Furthermore, eLSKSLAM progeny retained HSC potential, maintaining long-term multi-lineage reconstitution capacity in lethally ablated recipients. Increased hemopoietic cell proliferation was not MM contact dependent and could be recapitulated with media supplemented with two factors identified in MM-conditioned media: insulin-like growth factor binding protein-3 (IGFBP-3) and insulin-like growth factor-1 (IGF-1). We demonstrate that HSC express the receptor for IGF-1 and that IGF-1/IGFBP-3 induced increased hemopoietic cell proliferation can be blocked by an anti-IGF-1 neutralising antibody. However, co-cultures of 8N, 16N or 32N MM with eLSKSLAM showed that MM of individual ploidy did not significantly increase hemopoietic cell proliferation. Our data suggests that MM are an important component of the HSC niche and regulate hemopoietic cell proliferation through cytokine release.

SummaryHematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

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The existence of a bone marrow (BM) niche--the location in which hematopoietic stem cells (HSCs) reside--was proposed more than 30 years ago. Recent data suggest that the interaction of HSCs with cellular and extracellular components within the BM is critical for HSC regulation. The tracking of immunofluorescently labeled, prospectively isolated HSCs to and within the BM cavity allows the assessment of the regulatory processes involved in (1) homing, which involves transendothelial migration into the BM; (2) lodgment, including transmarrow migration through the extravascular space; and (3) BM reconstitution. Together, such analyses provide a better understanding of the cellular and extracellular components involved in the regulation of HSC quiescence and differentiation. Homing and lodgment of transplanted HSCs, the first critical steps in engraftment, involve multiple interactions between HSCs and the BM microenvironment. Herein, we describe a refined method of analyzing homing efficiency and spatial distribution of HSCs harvested from endosteal and/or central BM regions; we also review alternate methods. Using these techniques, microenvironment modifications within the recipient or surface protein-expression modifications on donor HSCs in animal models provide insights into components influencing the homing, lodgment, and engraftment processes.

Maintenance of hematopoietic stem cells (HSC) takes place in a highly specialized microenvironment within the bone marrow. Technological improvements, especially in the field of in vivo imaging, have helped unravel the complexity of the niche microenvironment and have completely changed the classical concept from what was previously believed to be a static supportive platform, to a dynamic microenvironment tightly regulating HSC homeostasis through the complex interplay between diverse cell types, secreted factors, extracellular matrix molecules, and the expression of different transmembrane receptors. To add to the complexity, non-protein based metabolites have also been recognized as a component of the bone marrow niche. The objective of this review is to discuss the current understanding on how the different extracellular matrix components of the niche regulate HSC fate, both during embryonic development and in adulthood. Special attention will be provided to the description of non-protein metabolites, such as lipids and metal ions, which contribute to the regulation of HSC behavior. J. Cell. Biochem. 118: 1984-1993, 2017. © 2017 Wiley Periodicals, Inc.

With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, damping of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.

Factor V (FV) and factor X (FX) activate and complex to form prothrombinase which subsequently cleaves prothrombin (PT), converting it to active thrombin. Thrombin cleaved osteopontin (tcOPN) contains a cryptic binding site for a 4 b 1 and a 9 b 1 integrins. We have previously shown that hematopoietic stem cells (HSC) bind to tcOPN via this site resulting in a decrease in their proliferation and differentiation. Therefore, tcOPN and the factors required for its generation are important components of the HSC niche. Herein we show mature megakaryocytes (MM, 8N) contain FV, FX, and PT mRNA and protein. Furthermore, we show 8N, 16N, 32N, and 64N MM all release the required factors to enable thrombin cleavage of OPN. Importantly, mice devoid of the myeloproliferative leukemia protein (Mpl), c-Mpl 2/2 mice, contain only approximately 10% of normal megakaryocyte numbers, showed significantly reduced FX and tcOPN protein levels in endosteal bone marrow (BM). In addition, WT hematopoietic progenitors and HSC showed reduced homing to the BM of c-Mpl 2/2 mice. This is the first report identifying MM as a key cellular component in the production of tcOPN in situ, allowing the BM microenvironment to self regulate HSC biology via tcOPN.

The α9β1 and α4β1 integrin subtypes are expressed on bone marrow haemopoietic stem cells and have important roles in stem cell regulation and trafficking. Although the roles of α4β1 integrin have been thoroughly investigated with respect to HSC function, the role of α9β1 integrin remains poorly characterised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to determine cell-associated protein localisation and activation, and to elucidate the mechanism of small molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent cell binding properties of a new fluorescent α9β1 integrin antagonist (R-BC154), which was based on a series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154 exhibits high nanomolar binding affinities to α9β1 integrin with potent cross-reactivity against α4β1 integrin under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differences in the binding kinetics between α9β1 and α4β1 integrins, which showed faster binding to α4β1 integrin relative to α9β1, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1) screening small molecule inhibitors of α9β1 and α4β1 integrins; (2) investigating the biochemical properties of α9β1 and α4β1 integrin binding and (3) investigating integrin expression and activation on defined cell phenotypes in vivo.