The rationale for purging of autologous acute myeloid leukemia (AML) grafts is to eradicate contaminating leukemic cells that might contribute to relapse. However, in vitro purging generally delays post-transplant hematopoietic recovery, thus increasing treatment-related complication rates. Theoretically, this prolonged aplasia might be shortened by the additional transplantation of ex vivo-generated progenitor cells. Therefore, we investigated whether nonleukemic progenitors could be expanded ex vivo from AML cell-contaminated CD34(+) peripheral blood progenitor cell (PBPC) preparations. Nonleukemic CD34(+)-selected PBPC and AML cells (Kasumi-1, KG-1, primary AML blasts) were cultured in cytokine-supplemented liquid culture for up to 19 days. Cells were used either unmanipulated or following in vitro purging with mafosfamide (30, 50, 75 microg/ml). Ex vivo-generated cells were assessed by flow cytometry, progenitor cell assays, and polymerase chain reaction. Without prior purging, ex vivo culture markedly amplified AML cells as well as nonleukemic CD34(+) PBPC (day 12: Kasumi-1, 18.5 +/- 0.6-fold; KG-1, 52.2 +/- 2.6-fold; CD34(+), 74.1 +/- 5.6-fold). Co-culture with leukemic cells did not affect CD34(+) cell growth and vice versa. Following in vitro purging, CD34(+) PBPC were expanded even at the highest mafosfamide dose (day 19: 25 +/- 15-fold), whereas leukemic cells were markedly depleted (approx. 1.5 log). Furthermore, normal colony-forming units (CFU) could be effectively recovered (day 19: 10 +/- 3.1% of prepurging input CFU), whereas CFU-L were depleted to undetectable levels in six of seven experiments. Finally, leukemic cells were undetectable following ex vivo co-culture of purged cells (CD34(+) PBPC plus 10% Kasumi-1 cells or primary blasts), but were clearly detectable without purging. Taken together, these data demonstrated that ex vivo expansion of normal progenitors from mafosfamide-purged AML cell-contaminated grafts might be feasible.