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Stem cells capable of differentiating to multiple lineages may be valuable for therapy. We report the isolation of human and rodent amniotic fluid-derived stem (AFS) cells that express embryonic and adult stem cell markers. Undifferentiated AFS cells expand extensively without feeders, double in 36 h and are not tumorigenic. Lines maintained for over 250 population doublings retained long telomeres and a normal karyotype. AFS cells are broadly multipotent. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. Examples of differentiated cells derived from human AFS cells and displaying specialized functions include neuronal lineage cells secreting the neurotransmitter L-glutamate or expressing G-protein-gated inwardly rectifying potassium channels, hepatic lineage cells producing urea, and osteogenic lineage cells forming tissue-engineered bone.