Preparation of mouse embryonic fibroblast feeder cells for human embryonic stem cell culture

INTRODUCTIONEmbryonic stem cells (ESCs) are derived from the inner cell mass of day 5-6 blastocysts. ESCs are pluripotent, meaning that they are able to differentiate into all derivatives of the three primary germ layers (ectoderm, endoderm, and mesoderm). In order to maintain the undifferentiated status of human ESCs (hESCs), feeder cells are used to provide both a suitable attachment substrate and critical soluble factors. Since the first hESC lines were established on mouse embryonic fibroblasts (MEFs), mitotically inactivated MEFs have commonly been used for supporting the culture of undifferentiated hESCs. Some previous studies suggest that MEFs may support hESC growth better than the human feeder cells typically isolated from post-natal tissues. This protocol describes a method for isolation and irradiation of MEFs for use in hESC culture.