Feeder-free growth of undifferentiated human embryonic stem cells

Previous studies have shown that maintenance of undifferentiated human embr yonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system i n which undifferentiated cells can be maintained for at least 130 populatio n doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off fe eders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a n ormal karyotype, stable proliferation rate, and high telomerase activity. S imilar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT alkaline phosphatase, and surface marker s including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintain ed without direct feeder contact formed teratomas in SCID/beige mice and di fferentiated in vitro into cells from all three germ layers. Thus, the cell s retain fundamental characteristics of hES cells in this culture system an d are suitable for scaleup production.