A family of plasma enzymes named amine oxidases contribute to a lack of survival of hematopoietic progenitor cells in vitro by oxidizing the amines released into the medium into cytotoxic and mutagenic hydrogen peroxide and aldehydes. This short-term toxicity manifests as low cloning efficiencies. Inhibitors of plasma amine oxidases prevent these toxic effects and increase cloning efficiencies for murine CFU-E CFU-Erythroid and CFU-Granulocyte/macrophage. These inhibitors can substitute for 2- mercaptoethanol in theCFU-E assay, providing an explanation for the stimulatory effects of thiols originally discovered by Iscove. However, these enzyme inhibitors are toxic to human cells, so other approaches are needed to eliminate amine oxidase toxicity. Serum-free media do not necessarily circumvent this problem, because many 'purified' proteins oradditives required for these media contain amine oxidases as contaminants. These enzymes also cause long-term toxicity, such as the crisis of spontaneous transformation in cultures of mouse embryo cells. These data suggest that amine oxidase toxicity is ubiquitous in vitro, and, furthermore, that the development of culture supplements devoid of amine oxidases may facilitate advances in the study and manipulation of stem cells ex vivo. In Phase I, we propose to determine which plasma amine oxidases cause toxicity. In Phase II, wewill develop technology to eliminate deleterious amine oxidases from the culture supplements, confirm that their elimination prevents short- and long-term toxicities, and apply this new cell culture technology toward the development of stem cell lines fromnormal and malignant human bone marrow, breast, prostate, and lymph node, including lymphomas.Such cell lines may prove valuable for carcinogenesis and mutagenesis studies, for in vitro assays for identifying new anti-cancer drugs, and for stabilizing the genome of hybridomas (currently cultured in 2-mercaptoethanol). The technology holds promise for a base from which many of the in vitro goals of """"""""Healthy People-2000"""""""" may be achieved.
