In vitro sensitivity of human hematopoietic progenitor cells to 4-hydroperoxycyclophosphamide

Protracted engraftment and prolonged thrombocytopenia remain problems when marrow purged with 4-hydroperoxy-cyclophosphamide (4-HC) is employed for autotransplants. Toxic effects of 4-HC on early progenitor and stem cells might play an important causative role. Surprisingly, few investigations have examined the effects of 4-HC on progenitor cells other than colony-forming units-granulocyte/macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E), and only one study could be found where 4-HC exposure was carried out on cells purified beyond the buffy coat stage. Since the cellular milieu, and in particular the red blood cell count, can ameliorate 4-HC toxicity, the suppressive effect of this agent on marrow stem cells might be underestimated. We therefore investigated the relative 4-HC sensitivity of different human bone marrow progenitor cells in vitro using partially purified adherent cell- and T lymphocyte-depleted bone marrow mononuclear cells (A-T-MNC). Cells were exposed to increasing doses (10 to 200 micrograms/mL) of 4-HC using a standard purging protocol established for bone marrow buffy coat cells. Effects on mixed (CFU-Mix), erythroid (CFU-E and BFU-E), granulocyte-macrophage (CFU-GM), and stromal cell (CFU-F) progenitors were determined. In addition, we examined the 4-HC sensitivity of megakaryocyte progenitors (CFU-Meg) since, to our knowledge, this has not been reported before. As expected, increasing doses of 4-HC led to progressive inhibition of hematopoietic colony formation. CFU-F, CFU-Mix, and CFU-Meg appeared most resistant to 4-HC exposure, while CFU-E, BFU-E, and CFU-GM appeared most sensitive. At doses over 100 micrograms/mL, the usual concentration recommended for purging of buffy coat cells, hematopoietic colony formation was completely inhibited. These data suggest that if more highly purified marrow cells are employed for purging, lower 4-HC doses may need to be used. They also suggest that the thrombocytopenia that frequently accompanies 4-HC purging is not likely due to loss of CFU-Meg.