Reproducible cloning assays for in vitro growth of canine hematopoietic progenitor cells and their potential applications in investigative hematotoxicity

A variety of in vitro cloning assays have been used for studying hematopoiesis in mice and human beings. However, these techniques have had limited use in dogs, a species used extensively as a model for hematopoietic research, particularly hematotoxicity. We have adopted cloning assays for in vitro growth of canine colony-forming unit-erythroid (CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitor cells, using modified microplasma clot and soft agar culture systems respectively. Marrow mononuclear cells separated by density-gradient centrifugation were added to the aforementioned culture systems. Erythroid colonies were stimulated with sheep plasma erythropoietin and incubated at 37 C in 5% CO2 for 2 days. The CFU-E colonies were fixed with 5% glutaraldehyde, stained with benzidine, counted, and expressed as a mean of 8 replicates. The CFU-GM colonies were stimulated with pooled serum from endotoxin-treated dogs and incubated for 8 days at 37 C in 10% CO2. Using an inverted microscope, the CFU-GM colonies were counted and expressed as a mean of 6 replicates. The number of colonies was proportional to the plated cell concentrations. The addition of 10% autologous serum to CFU-GM cultures increased the number of colonies by 80 to 100%, but markedly reduced the size and number of CFU-E colonies. The marrow cloning capacity among dogs of comparable age was similar, and little variation was noticed when bone marrow cells from the same dogs were cultured repeatedly over a period of 3 to 4 months. We concluded that these cloning assays are fast, reliable, and reproducible and that they allow quantitative determination of canine hematopoietic progenitor cells. The assays may be useful in screening the hematotoxic potential of various therapeutic agents and are particularly suited for studying the pathogenetic mechanisms of drug-induced blood disorders and their reversibility.