Previous work has revealed that a CD34-negative, fibroblast-like cell is the earliest hematopoietic progenitor population in the bone marrow. 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 interleukin-6(IL-6)-mediated plastic adherence. We isolated peripheral blood-derived mononuclear cells from mice and dogs and established in vitro a fibroblast-like, adherently growing cell layer. Cells had to be immortalized for cellular cloning. Monoclonal fibroblast-like cell lines were established, and the surface expression of early stem cell markers was determined by flow cytometry. Clones were predominantly negative for CD34, but expressed the stem cell factor receptor c-kit. Irradiated mice and dogs were successfully transplanted with their autologous and syngeneic CD34-negative, fibroblast-like cell clones, respectively. After stem cell transplantation fluorescence in situ hybridization or green fluorescence expression revealed the presence of donor hematopoietic cells in the recipient bone marrow. Immunohistochemistry for the expression of various surface antigens showed the presence of the phenotype of the transplanted stem cell clone along the bone spicules in the marrow cavity, giving rise to hematopoietic progenitor cells of all lineages. In addition, CD34-negative, c-kit-positive hematopoietic stem cells also expressed osteocalcin, suggesting a role in osteogenesis. In summary, we have shown that a CD34-negative, c-kit-positive fibroblast-like cell clone represents the earliest hematopoietic and repopulating stem cell population. CD34-negative, fibroblast-like hematopoietic stem cells can also be isolated from human peripheral blood-derived mononuclear cells. A primary fibroblast-like cell culture can be established within 3–4 weeks. Fibroblast-like hematopoietic stem cells are also efficient targets for somatic gene transfer due to the highly efficient transduction efficacy.

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