Propagation of human embryonic stem cells on human feeder cells.

Human embryonic stem (hES) cell lines are usually derived and propagated on inactivated murine embryonic fibroblast (MEF) feeders. The use of MEFs and culture ingredients of animal origin for hES cell support increases the risk of cross-contamination of the hES cells with infectious animal agents from the MEFs and animal-based culture medium. This thus makes such hES cells lines undesirable for clinical application. This chapter describes several protocols used in the propagation of hES cells on human fibroblast feeder cells. Two culture methods, the bulk enzymatic culture protocol and the microdissection "cut and paste" protocol are described. Only certain human fetal and adult fibroblast feeders support hES cell growth. Methods for the characterization of pluripotent undifferentiated hES cells grown on human feeders including cell surface marker staining and real-time polymerase chain reaction are also described.