Influence of erythropoietin plus granulocytecolony stimulatin factor on in-vitro migration of progenitors from murine bone marrow

In an effort to develop a relevant in-viîro model of cytokine-induced mobilization of hematopoietic progenitors from bone marrow, a murine system was designed utilizing transwells (Costar, Cambridge, MA). The murine stromal cell line MS5 was grown to confluence on polyester or polycarbonate transwell membranes with 3 micron pores. A femoral marrow plug from an intact Balb/C mouse was placed on an MS5 covered transwell and seated in a media-containing well. Six such transwells were used for each experiment. Twenty four hours later, all non-adherent cells which had migrated to the wells were harvested and the transwells moved to new wells containing media plus cytokines in concentrations that mobilize well in vivo [500 U/kg erythropoietin (EPO) and 1Sjig/kg granulocyte-colony stimulating factor (G-CSF)] where they remained for a further 24 hours. Then, non-adherent cells were harvested from the wells and the transwells were moved to new wells containing media and cytokines and remained there for 48 hours. Finally, cells were harvested from the wells and the transwells were moved again to new wells containing cytokine and media where they remained for 72 hours. Then, 7 days after the marrow plug was placed in the transwell, all non-adherent cells were harvested from the wells and all adherent and nonadherent cells were harvested from the transwells. Each time cells were harvested, they were counted and assayed in triplicate for GM-CFC using a standard methylcellulose culture system. Low numbers of cells and GM-CFCs were present in the wells after the transwells had been seated 24 hours in media alone. After 24 and 48 hours in media plus cytokines, an increased number of cells had migrated to the wells, but they included few to no assayable GM-CFCs. After being seated in the wells containing cytokines and media for 72 hours, the number of migrated cells was approximately equivalent to that found after 34 and 48 hours, but the number of GM-CFCs was increased approximately one log. The number of GMCFCs remaining in the transwells after 7 days was approximately 20% of the total number of GM-CFCs originating from the marrow plug. An in-vitro murine system of cytokineinduced migration of hematopoietic progenitors from bone marrow has been developed. This system may allow dissection of mobilization differences between tumor cells and progenitor cells.