CD34-negative hematopoietic stem cells isolated from human peripheral blood cells as ultimate precursors of hematopoietic progenitors

Background: The earliest hematopoietic stem cell is still not vet identified. From experiments in dogs there is evidence that the earliest hematopoietic stem cell is a CD34(-) fibroblast-like cell which can be isolated from bone marrow stromal cultures, In this set of experiments we wanted to identify such an early CD34- fibroblast-like cell population from human peripheral blood. Material and Methods: Peripheral blood mononuclear cells (PBMNC) were isolated from healthy volunteer donors and incubated with interleukin-6(IL-6)-containing medium in tissue culture flasks. After establishing an almost confluent adherent layer of fibroblast-like cells which attach to the plastic surface, the cells were stimulated with various growth factors. The emerging nonadherent cells as well as the adherent cells were applied to a colony-forming unit (CFU) assay in semisolid agar and to long-term culture-initiating cell (LTC-IC) assay. Results: CD34- adherently growing cells could be isolated from human peripheral blood by IL-6-mediated plastic adherence. These cells grow as fibroblast-like cells in culture and differentiation can be induced by stem cell factor (SCF), while IL-6 induces proliferation of these cells. While differentiating, CD34- fibroblast-like cells lose the ability to row adherently and start to express CD34 and eventually HLA-DR, The differentiating CD34+ cells are also capable of producing CFU and LTC-IC. Conclusion: This set of experiments provides evidence that the earliest identifiable hematopoietic stem cell is a CD34+ adherently growing fibroblast-like cell which is part of the marrow microenvironment in vivo and can differentiate into hematopoietic progenitors upon appropriate growth factor signaling. This kind of early stem cell can also be isolated from PBMNC.