The ex vivo expansion of feline marrow cells leads to increased numbers of BFU-E and CFU-GM but a loss of reconstituting ability

Some studies in mice suggest that hematopoietic stem cells can be maintained and possibly expanded ex vivo. As there is a paucity of data from larger animals, we have studied hematologic reconstitution following autologous marrow transplantation in cats, Transplantation of very low density marrow cells (<1.050 g/ml), termed "1050 cells," at 2 x 10(5) cells/kg leads to rapid hematopoietic recovery (granulocytes >200/mu l by day 20 +/- 2 and platelets >50 x 10(3)/mu l by day 21 +/- 3). Recovery rates are comparable when 1-2 x 10(7) nucleated marrow cells/kg are infused, suggesting that reconstituting cells are enriched 50- to 100-fold in the 1050 cell preparation. To explore if the numbers of reconstituting cells could be expanded ex vivo, 1050 cells were cultured in the presence of 5 ng/ml recombinant human interleukin 1 beta, 10 ng/ml recombinant canine (rc)G-CSF, 2 U/ml rHu erythropoietin, and 5 ng/ml rc stem cell factor. Maximum numbers of BFU-E and colony-forming units-granulocyte/macrophage (CFU-GM) were generated at day 6, However, when 10(6) 1050 cells/kg (5x that needed for hematologic recovery) mere cultured for six days and all resulting cells infused into irradiated donor animals, two of nine (22%) engrafted. Even when flt3 Ligand (100 ng/ml) was added to cultures, only two of five animals (40%) engrafted (p = NS versus studies without flt3 ligand). These data confirm that BFU-E and CFU-GM provide inaccurate estimates of reconstituting cells and demonstrate that the number or function of feline reconstituting cells is impaired by in vitro culture with cytokines.