Human embryonic stem cells (HES) hold great potential for regenerative medicine because of their ability to differentiate to any cell type. However, a limitation is that HES cells require a feeder layer to stay undifferentiated. Routinely, mouse embryonic fibroblast is used. However, for therapeutic applications, contamination with mouse cells may be considered unacceptable. In this study, we evaluated three commercially available human foreskin feeder (HF) lines for their ability to support HES cell growth in media supplemented with serum or serum replacer. HES cells on HF in serum replacer-supplemented media were cultured for >30 passages. They remained undifferentiated, maintained a normal karyotype, and continued to be positive for the pluripotent markers Oct-4, SOX-2, SSEA-4, GCTM-2, Tra-1-60, Tra-1-81, and alkaline phosphatase. In vivo, HES cells formed teratomas in SCID mouse models that represent the three embryonic germ layers. In contrast, HES cells cultured on HF in serum-supplemented media differentiated after three passages. Morphologically, the cells became cystic with a loss of intracellular Oct-4. We have successfully adapted and cultured undifferentiated HES cells on three human feeder lines for >30 passages. No difficulties were observed with the exception of serum in the media. This study reveals a safe and accessible source for feeders for HES cell research and potential therapeutic applications. (C) 2004 Wiley Periodicals, Inc.