Previous work had revealed that a CD34- fibroblast-like cell is the earliest hematopoietic progenitor population. This cell type is able to differentiate into hematopoietic progeny of all lineages and circulates in the peripheral blood, from where it can be isolated by IL-6-mediated plastic adherence. We isolated peripheral blood-derived mononuclear cells (MNC) from male CBA mice and established in vitro a fibroblast-like, adherent growing cell layer. Cells were immortalized by SV-40 transfection for cellular cloning. Monoclonal fibroblast-like cell clones were established, and the surface expression of early stem cell markers was determined by flow cytometry. Clones were CD34-, Sca-1+, Thy-1(low), and c-kit+. Lethally irradiated female CBA mice were successfully transplanted with a fibroblast-like cell clone, R-M26/2-1. After syngeneic transplantation, peripheral blood counts were back to normal in transplanted mice on days 15-20, and fluorescence in situ hybridization (FISH) revealed the sole presence of male hematopoietic cells in the BM of female recipients at weeks 7, 9, 11, and 16 after transplantation. Immunohistochemistry for the expression of CD34, Sca-1, Thy-1, and c-kit showed the presence of the phenotype of the transplanted stem cell clone along the bone spicules in the marrow cavity, giving rise to HPC of all lineages. In summary, we have shown that a CD34-, Sca-1+, Thy-1(low), and c-kit+ fibroblast-like cell is consistent with the phenotype of the earliest hematopoietic and repopulating stem cell and can be isolated from peripheral blood cells.
