Long-term “in vitro” proliferating mouse hematopoietic progenitor cell lines

Abstract

Long-term proliferating hematopoietic progenitor cell lines have been established from mouse bone marrow in tissue cultures on the M-CSF-deficient stromal cell line OP9. In the presence of stem cell factor (SCF), thrombopoietin, IL-3 and IL-6 pluripotent hematopoietic stem cells (pHSC) initiate proliferation. For 2–3 weeks they maintain long-term reconstitution capacity, as tested in adoptive transfer experiments into sublethally irradiated hosts, but later loose this capacity. Transfection with HOXB4 stabilises the pluripotency and long-term reconstitution capacity of these pHSC-like cell lines. Transfer into media containing SCF and FLT3L, the ligand for flt3, develops cell lines with myelopoietic and lymphopoietic potencies, reconstituting hosts with a wave of short-term reconstitutions of these cell lineages. Subsequent transfer into cultures containing SCF, FLT3L and IL-7 generates lines with lymphoid reconstitution capacities, i.e. able to develop T-lineage, B-lineage and NK-lineage cells. Again, this multi-lymphoid lineage developmental capacity is lost within 2 weeks, so that the remaining, proliferating cells generate B-lineage cells only, when induced to differentiate. These cell lines become capable to proliferate in IL-7 alone and now resemble pre BI-Type cell lines, as those previously isolated from fetal liver. Hence such preBI cell lines can be generated by a stepwise alteration of the cytokine milieu in culture from pHSC but intermediate differentiation stages still need to be stabilized in attempts to establish long-term proliferating cell lines at different stages of hematopoietic development.