FIBROBLASTS RETROVIRALLY TRANSFECTED WITH THE HUMAN IL-3 GENE INITIATE AND SUSTAIN MULTILINEAGE HUMAN HEMATOPOIESIS IN SCID MICE - COMPARISON OF CD34-ENRICHED VS CD34-ENRICHED AND IN-VITRO EXPANDED GRAFTS

Peripheral blood progenitor cells (PBPCs) obtained from cytapheresis p roducts (CPs) of tumor patients undergoing mobilizing chemotherapy for PBPC support and dose-intensified anticancer chemotherapy initiate mu ltilineage human hematopoiesis after intraperitoneal (i.p.) transplant ation into young severe combined immunodeficient (SCID) mice, The engr aftment process was significantly accelerated by subcutaneous cotransp lants of a rat fibroblast cell line stably transfected with a retrovir al vector carrying the human interleukin-3 (hIL-3) gene and producing sustained in vivo levels of circulating human IL-3 over a prolonged pe riod of time. These cotransplants were found to provide a suitable mic roenvironment for i.p. transplanted CD34-positive cells separated from PBPC preparations using immuno-magnetic beads. Flow cytometry analysi s and immuno-cytology revealed that selected PB CD34(+) cells, more th an 90% pure, were capable of initiating and sustaining a productive mu ltilineage hematopoiesis preferentially within the hIL-3-secreting cot ransplants followed by release of mature human cells into the circulat ion, spleen and thymus. The percentages of human cells found in hIL-3 cotransplants 8 weeks post-transplantation (p.t.) were generally highe r than those measured after transplantation of complete CP mononuclear cells containing comparable doses of CD34-positive cells, When select ed PB CD34(+) cells that were expanded ex vivo with combinations of hu man hematopoietic growth factors including the c-kit ligand (KL), inte rleukin (IL)-1 beta, IL-3, IL-6, erythropoietin (EPO) and granulocyte- macrophage colony-stimulating factor (GM-CSF) for 14 days were grafted to cotransplant-carrying SCID mice, a considerable loss of their prol iferative potential was observed regardless of the HGF combination use d, When experiments with grafts of selected PBPC were compared to thos e performed with selected/expanded PBPC on a per CD34(+) cell basis th e results revealed that over a dose range of 0.3 to 1.0 x 10(6) cells/ graft the in vivo proliferative capacity of expanded cells was reduced by a factor of 2 to 3.