BACKGROUND: Although ex vivo expansion of bone marrow (BM) cells has been proposed as an effective method for the early recovery from pancytopenia in patients with bone marrow stem cell transplantation (BMT), the expansion potential and the long-term reconstitution capability of such BM cells is still controversial. We describe here a multiple cytokine medium (MCM) containing major hematopoietic stimulation factors and conditioned medium from PHA-stimulated murine spleen cells that permits the expansion of BM cells with long-term hematopoietic reconstitution capacity. MATERIAL/METHODS: Male murine BM cells were expanded in MCM for 4 to 14 days and injected into lethally irradiated syngeneic female mice. The mice were maintained for 18 months after transplantation for evaluation of hematopoietic reconstitution. RESULTS: The expanded cells contained pluripotent hematopoietic stem cells and lineage committed progenitors as well as terminally differentiated cells. They permitted full recovery of lethally irradiated mice in both early and late stages in same numbers equivalent to that of unexpanded cells. More than 80% of the progenitor cells were donor originated after 18 months. Expanded cells were able to be transduced with a retroviral vector expressing Beta-galactosidase, and continued to express the marker following BMT. CONCLUSIONS: With the use of MCM, the quantity of donor cells from BM and other sources might be greatly reduced. Ex vivo expanded BM cells might also facilitate gene manipulation in vitro by retroviral vectors.

Keywords: Bone Marrow Transplantation - physiology, Bone Marrow Cells - physiology, Bone Marrow Transplantation - physiology, Cell Division - drug effects, Colony-Forming Units Assay, Cytokines - pharmacology, DNA Primers, Hematopoiesis - physiology, Phytohemagglutinins - pharmacology, Polymerase Chain Reaction, Spleen - drug effects, Y Chromosome