No abstract

Herein, we investigated the role of VEGF signaling in the earliest events in vasculogenesis and found that it exerts critical effects shortly after mesodermal cells form by gastrulation. We showed that VEGF treatment of embryos caused an increase in the population of newly gastrulated mesodermal (NGM) cells that express the transcription factor TAL1. This increase in TAL1-positive cells was attributed to VEGF induction of VEGF receptor-2 (Flk1)-positive NGM cells that would normally not have been induced due to the limited availability of VEGF in the NGM. Evidence that VEGF-mediated induction of NGM cells is relevant to the endothelial lineage is the finding that induced TAL1-positive cells in the NGM formed ectopic structures whose cells exhibited characteristics of endothelial cells, including the ability to integrate into the vascular network and express the QH1 antigen. Finally, we showed that VEGF-induced TAL1 expression in the NGM which resulted in the formation of ectopic structures was mediated by Flk1 but not Flt1 signaling. In summary, we have established that VEGF signaling is critical to allocation of NGM to the endothelial lineage.

Recent studies indicate that hematopoietic stem cells (HSCs) are capable of reconstituting a number of non-hematopoietic organs and tissues. To define the potential of the HSC precisely, we initiated transplantation studies of a clonal population of cells derived from a single HSC. We used the bone marrow (BM) of transgenic enhanced green fluorescent protein (EGFP) mice as the source of donor HSCs. We demonstrated the HSC origin of glomerular mesangial cells (Masuya et al, Blood 101: 2215, 2003) and brain microglial cells and pericyte-like perivascular cells (Hess et al, Exp Neurol 186: 134, 2004). These observations and the fact that glomerular mesangial cells and pericytes are considered myofibroblasts suggested that fibroblasts are also derived from HSCs. In this abstract, we present evidence for HSC origin of fibroblasts and their precursors, BM fibroblast colony-forming units (CFU-F) and peripheral blood (PB) fibrocytes. Lin−Sca-1+c-Kit+CD34− cells from the BM of adult EGFP mice were individually sorted into 96 well Corning plates and cultured for 7 days in the presence of Steel factor and IL-11 or Steel factor and G-CSF. Viable clones consisting of fewer than 20 cells were individually transplanted into lethally irradiated mice. EGFP+ mononuclear cells were sorted from the BM cells of recipients showing high-level, multilineage hematopoietic reconstitution and assayed for CFU-F in Retronectin-treated tissue culture plates. Colonies consisting of more than 50 adherent cells were all EGFP+. The majority of the cells comprising the colonies were fibroblast-like, exhibited spindle-shaped or polygonal cytoplasm and had clear, ovoid nuclei. Flow cytometric analyses revealed that these cells expressed collagen-1 and discoidin domain receptor 2 (DDR2) and exhibited a decreased intensity of CD45. RT-PCR analysis of these cells revealed the presence of mRNA for procollagen 1 alpha-1, vimentin, fibronectin and DDR2. Next we analyzed the PB for donor origin fibrocytes, a fibroblast-like cell type that expresses both fibroblastic and hematopoietic phenotypes in culture. When nucleated PB cells from clonally engrafted mice were cultured on fibronectin-coated dishes, proliferation of EGFP+ fibroblast-like cells was detected. Only one-third of the EGFP+ cells expressed CD45 and most of the EGFP+ cells expressed both collagen-I and DDR2. Similar results were obtained with EGFP+ cells from mice transplanted with 100 uncultured Lin−Sca-1+c-Kit+CD34− cells or 1x106 BM nucleated cells. These studies excluded the effects of short-term culture on HSC differentiation and established the HSC origin of CFU-F and fibrocytes. Classic studies of CFU-F by Friedenstein and others have led to the general belief that mesenchymal stem cells (MSCs), rather than HSCs, generate a number of tissues including adipocytes, osteoblasts, chondrocytes, myocytes and vascular endothelial cells. Here we unequivocally demonstrated that CFU-F are of HSC origin, warranting a re-evaluation of the relationship between HSCs and MSCs.