Survival of Highly Proliferative Colony Forming Cells after Treatment of Bone Marrow Cells with 4-Hydroperoxycyclophosphamide

We investigated the in vitro effects of 4-hydroperoxycyclophosphamide (4-HC) on human hemopoietic stem cells. Marrow cells were exposed to 4-HC and then assayed for mixed (CFU-GEMM), erythroid (BFU-E) megakaryocyte (CFU-M), and granulocyte-macrophage (CFU-GM) colony forming cells. We found that highly proliferative colony forming cells, especially CFU-GEMM and BFU-E, were relatively spared by 4-HC treatment. One third of the surviving progenitors formed large colonies, some of which contained more than 50, 000 cells. By sequential examination of the formation of these large colonies, we found immature colonies consisting of blasts at the early stage of culture. The morphology of these “blast cell colonies” in situ was arbitrarily classified into four types. Among them were the blast cell colonies consisting of the individual cells that were dispersed and had a few granules within the cytoplasm (type A); these cells finally formed very large colonies on day 22 of culture. Approximately 70% of the single cells derived from type A blast cell colonies produced secondary colonies consisting of erythroblasts, macrophages, eosinophils, and/or basophils. These results show that the blast cells in type A colonies have a highly proliferative capacity. The availability of a highly enriched population of primitive hemopoietic progenitors will provide us with a unique opportunity to study the interaction between a single stem cell and purified hemopoietic factors.