A critical regulatory role of hematopoietic stem cell (HSC) vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT ligand. However, endothelial-derived KIT ligand is expressed in both a soluble and membrane-bound form and not unique to bone marrow niches, and it is also systemically distributed through the circulatory system. Here, we confirm that upon deletion of both the soluble and membrane-bound forms of endothelial-derived KIT ligand, HSCs are reduced in mouse bone marrow. However, the deletion of endothelial-derived KIT ligand was also accompanied by reduced soluble KIT ligand levels in the blood, precluding any conclusion as to whether the reduction in HSC numbers reflects reduced endothelial expression of KIT ligand within HSC niches, elsewhere in the bone marrow, and/or systemic soluble KIT ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion, specifically of the membrane-bound form of KIT ligand, also reduced systemic levels of soluble KIT ligand, although with no effect on stem cell numbers, implicating an HSC regulatory role primarily of soluble rather than membrane KIT ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT ligand, HSCs were unaffected in KIT ligand deleted bones implanted into mice with normal systemic levels of soluble KIT ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow.

Despite much work studying ex vivomultipotent stromal cells ( MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP(+) MSCs in various organs. In vivo, EGFP(+) mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP(+) cells exhibited a perivascular distribution pattern. EGFP(+) cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP(+) specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP(+) population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD–driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

Thymic T cell development is initiated from bone-marrow-derived multi potent thymus-seeding progenitors. During the early stages of thymocyte differentiation, progenitors become T cell restricted. However, the cellular environments supporting these critical initial stages of T cell development within the thymic cortex are not known. Here we use the dependence of early, c-Kit-expressing thymic progenitors on Kit ligand (KitL) to show that CD4(-)CD8(-)c-Kit(+)CD25(-) DN1-stage progenitors associate with, and depend on, the membrane-bound form of KitL (mKitL) provided by a cortex-specific KitL-expressing vascular endothelial cell (VEC) population. In contrast, the subsequent CD4(-)CD8(-)c-Kit(+)CD25(+) DN2-stage progenitors associate selectively with cortical thymic epithelial cells (cTECs) and depend on cTEC-presented mKitL. These results show that the dynamic process of early thymic progenitor differentiation is paralleled by migration-dependent change to the supporting niche, and identify VECs as a thymic niche cell, with mKitL as a critical ligand.

The identification of a functionally distinct subset of haematopoietic stem cells (HSCs) that is primed for platelet-specific gene expression is described; the cells frequently have long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs, and may enable the design of therapies for enhancing platelet reconstitution.

From Urist’s studies (1965) to the following observations by Sampath and Reddi (1981) on the role of BPM morphogenetic protein and, more particularly, BPMs/ Ops in the complex mechanism of bone regeneration to recent studies (Solheim, 1998) on the properties of the single elements belonging to the superfamily of AGF of platlet derivation to activate the specific receptors of stem cells, considerable progress has been made in research and clinical application. The non-specificity of the present evaluation methods of patient outcome prevent us from singling out which element has activated the osteogenetic process, and when and which one has affected the course favourably. In order to answer all these questions, we have carried out a prospective study between autologous AGF and stem cell application in orthopaedics and the ex vivo evaluation in vitro of platelet activation of osteoblasts. From March 2002 to January 2004, we treated 21 cases of osteolysis (in mobilisations due to pros-thesis of hip and knee, necrosis of bone head femur, pseudoarthrosis of the humerus and ulna, and aneurysmal relapsing cyst). The procedure consisted in using stem cells+AGF+autologous bone graft (two cases) or bank bone graft (13 cases)-or hydroxyapatite (two cases)+autologous fibrin glue and at the same time, in vitro culture of the patient’s osteoblasts+AGF. Among the AGF, PDGF is an important mitogenous factor. Among the isoforms of PDGF, the PDGF-AA and PDGF-AB are stored in alpha –granules and released when platelets are activated. PDGF-AA is preferentially secreted by osteoblasts and fibroblasts. PDGF-AA and PDGF-AB were measured in supernatants of osteoblasts in culture media, activated platelet culture media, osteoblasts plus activated platelet culture media at day 0, day +4, after adding platelet and day +8. At the end a cytological test was performed. The study has proved that the PDGF-AA increases remarkably after adding platelet gel to the osteoblasts, more than in control cultures which are lacking in platelet gel, which might mean the platlets activate the osteoblast and activate the osteogenetic process. The use of autologous fibrin glue enriched in AGF has demonstrated a better recovery of the tissue scar, as well as emathic save and better stop of grafts. The range of cases is not homogeneous and prevents us from drawing any statistical conclusions, but the quick bone recovery leads to great satisfaction both for patients and their physicians.