INTRODUCTIONThe potential of human embryonic stem cells (hESCs) to differentiate into diverse cell types of all major tissue types has raised hope that hESCs can be used for novel cell-based therapies as well as fundamental studies of cell lineage differentiation. In order to support the growth of hESCs, mouse embryonic fibroblasts (MEFs), typically isolated from E13-E14 mouse embryos and between passages three and seven, are routinely used as feeder cells. Feeder density is a critical determinant of successful hESC culture. In the presence of too few feeder cells, hESCs may not maintain self-renewal and pluripotency properties; they may also fail to attach to feeder cells appropriately. At excessively high density, feeder cells may detach from the plate such that hESC colonies will be lost. Here we describe an efficient method for culturing and passaging hESCs by enzyme (collagenase) for culture on irradiated MEFs. The protocol can be used for routine hESC culture for basic research.