Although recent data suggests that osteoblasts play a key role within the hematopoietic stem cell (HSC) niche, the mechanisms underpinning this remain to be fully defined. The studies described herein examine the role in hematopoiesis of Osteopontin (Opn), a multidomain, phosphorylated glycoprotein, synthesized by osteoblasts, with well-described roles in cell adhesion, inflammatory responses, angiogenesis, and tumor metastasis. We demonstrate a previously unrecognized IntroductionHematopoietic stem cell (HSC) engraftment is a multistep process, involving homing, transmarrow migration (TMM), and lodgment within a bone marrow (BM) niche. Homing is the specific recruitment of HSCs to the BM and involves the recognition of HSCs by the BM microvascular endothelium and transendothelial cell migration into the hematopoietic space. Lodgment is defined as the selective migration of HSCs to a suitable niche within the extravascular compartment. In comparison to homing, very little is known about molecules that regulate HSC lodgment and, moreover, the retention of HSCs within these distinct anatomical locations.In accord with the stem cell niche model proposed by Schofield, 1 recent studies within our laboratory demonstrate that HSCs actively migrate toward and reside within the endosteal region at the bone and BM interface. 2,3 This concept is supported by studies reported by Calvi et al, 4 Zhang et al, 5 and Arai et al, 6 which highlight the importance of direct contact and interactions between HSCs and osteoblasts at the endosteal surface in the regulation of HSC proliferation. Further evidence that osteoblasts directly regulate hematopoiesis is provided by studies in which conditional ablation of osteoblasts results in significant reduction of marrow hematopoiesis, 7 although the factors responsible for this profound effect remain to be determined. In addition, in vitro evidence indicates that osteoblastic cells can expand HSC numbers 8 and, when cotransplanted with HSCs, can improve engraftment. 9 Collectively, these findings suggest that osteoblasts are a key cell type within the HSC niche and that molecules expressed by these cells may have previously unrecognized roles in regulating hematopoiesis.One molecule that shows high levels of expression in osteoblasts cells lining bone trabeculae is Osteopontin (Opn), 10 an observation that is not unexpected given its well-described role as a key regulator of bone homeostasis. 11 Opn is a multidomain, phosphorylated glycoprotein synthesized by many cell types and involved in many physiologic and pathologic processes, including cell adhesion, 12 angiogenesis, 13 apoptosis, inflammatory responses, and tumor metastasis. 14 Physiologically, phosphorylation, glycosylation, and cleavage of Opn result in molecular mass variants, ranging from 25 to 75 kDa. The different effects that Opn elicit are attributable to its multiple receptors, binding sites, and its various forms. 15 One of the major serine proteases to cleave Opn is thrombin, giving rise to a 24-kDa and a 45-kDa fragm...

The role of lung epithelial stem cells in maintenance and repair of the adult lung is ill-defined, and their identity remains contentious because of the lack of definitive markers for their prospective isolation and the absence of clonogenic assays able to measure their stem/progenitor cell potential. In this study, we show that replication of epithelial-mesenchymal interactions in a previously undescribed matrigel-based clonogenic assay enables the identification of lung epithelial stem/progenitor cells by their colonyforming potential in vitro. We describe a population of EpCAM hi CD49f pos CD104 pos CD24 low epithelial cfus that generate colonies comprising airway, alveolar, or mixed lung epithelial cell lineages when cocultured with EpCAM neg Sca-1 pos lung mesenchymal cells. We show that soluble fibroblast growth factor-10 and hepatocyte growth factor partially replace the requirement for mesenchymal support of epithelial colony formation, allowing clonal passaging and demonstration of their capacity for self-renewal. These data support a model in which the adult mouse lung contains a minor population of multipotent epithelial stem/progenitor cells with the capacity for self-renewal and whose descendants give rise to airway and alveolar epithelial cell lineages in vitro.colony-forming assay | lung epithelium | lineage specificity | differentiation | EpCAM

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The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 vessels from which RUNX1C blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

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.

Originally identified as a marker specifying murine hematopoietic stem cells, the Sca-1 antigen has since been shown to be differentially expressed by candidate stem cells in tissues including vascular endothelium, skeletal muscle, mammary gland, and prostate of adult mice. In the adult murine lung, Sca-1 has previously been identified as a selectable marker for the isolation of candidate nonhematopoietic (CD45 Sca-1 1 CD34 1 cell phenotype does define endogenous clonogenic progenitor cells in the adult murine lung, our data indicate that these progenitors are predominantly representative of mesenchymal cell lineages, and highlights the pressing need for the identification of alternative markers and robust functional assays for the identification and characterization of epithelial and fibroblastic stem and progenitor cell populations in the adult lung. STEM CELLS 2009;27:623-633 Disclosure of potential conflicts of interest is found at the end of this article.

Despite the fact that many hypoxia-inducible genes are important in hematopoiesis, the spatial distribution of oxygen in the bone marrow (BM) has not previously been explored in vivo. Using the hypoxia bioprobe pimonidazole, we showed by confocal laser scanning microscopy that the endosteum at the bone-BM interface is hypoxic, with constitutive expression of hypoxia-inducible transcription factor-1␣ (HIF-1␣) protein in steady-state mice. Interestingly, at the peak of hematopoietic stem and progenitor cell (HSPC) mobilization induced by either granulocyte colony-stimulating factor or cyclophosphamide, hypoxic areas expand through the central BM. Furthermore, we found that HSPC mobilization leads to increased levels of HIF-1␣ protein and increased expression of vascular endothelial growth factor A (VEGF-A) mRNA throughout the BM, with an accumulation of VEGF-A protein in BM endothelial sinuses. VEGF-A is a cytokine known to induce stem cell mobilization, vasodilatation, and vascular permeability in vivo. We therefore propose that the expansion in myeloid progenitors that occurs during mobilization depletes the BM hematopoietic microenvironment of O 2 , leading to local hypoxia, stabilization of HIF-1␣ transcription factor in BM cells, increased transcription of VEGF-A, and accumulation of VEGF-A protein on BM sinuses that increases vascular permeability.

We have studied the properties of a glial progenitor cell from 7‐day‐old rat optic nerve that differentiates in vitro into an oligodendrocyte if cultured in serum‐free medium and into an astrocyte if cultured in foetal calf serum (FCS). Using galactocerebroside as a marker of oligodendrocyte differentiation and glial fibrillary acidic protein as a marker of astrocyte differentiation, we show that the acquisition of these marker molecules occurs rapidly in culture and requires both RNA and protein synthesis. We provide evidence that the effect of FCS on the development of the glial progenitor cell is not due to its influence on cell‐substrate adherence or actin filament organization and is not mimicked by an increase in intracellular cyclic AMP, cyclic GMP or pH. The progenitor cell contains vimentin filaments and retains them on becoming an astrocyte but loses them on becoming an oligodendrocyte. Most importantly, we show that the choice of developmental pathway taken by the bipotential glial progenitor cells in culture is reversible for 1‐2 days and then becomes fixed, at least under the conditions we studied.