Differential adherence of murine hematopoietic stem cell subsets to fibronectin

Growth and differentiation of hematopoietic stem cells occur in close contact with the cells and extracellular-matrix (ECM) proteins of the hematopoietic microenvironment. We observed the role of fibronectin, a matrix glycoprotein proposed to be involved in the attachment of hematopoietic cells and in the binding of stem cell subsets to bone marrow stroma, for this study. Murine bone marrow cells (BMC) were allowed to adhere to surfaces coated with human plasma fibronectin. Using the cobblestone-area-forming cells (CAFC) assay, adherent and nonadherent cell fractions were tested for their quantity of primitive and less primitive stem cell subsets. The CAFC assay is based on a time-dependent clone formation in pre-established, bone marrow-derived, irradiated stromal layers under limiting dilution conditions, and it allows in vitro enumeration of day-12 colony-forming unit-spleen (CFU-S12) (CAFC-10) and more primitive cells with long-term repopulating abilities (LTRA) (CAFC-28/35). We observed that the majority of primitive CAFC-28/35 adhered to fibronectin, while only a minority of CFU-S-like CAFC-10 did. The adherence of primitive stem cells to fibronectin could partially be blocked by high molar concentrations of oligopeptides containing the essential amino acid sequence of the central cell-binding domain of fibronectin, RGD. Adherence of the small subpopulation of CAFC-10 to fibronectin could almost entirely be prevented by oligopeptides organized in a specific fashion. These data suggest a role for RGD-binding integrins in the adherence of hematopoietic stem cells.