Culture of human embryonic stem cells

The successful culture and maintenance of human embryonic stem cells (HESCs) offers great promise for furthering our basic understanding of many regulatory and developmental processes, and for developing effective clinical applications for treating, even correcting, genetic disorders. Realizing these potentials demands a better understanding of the fundamental aspects of HESCs in vitro, including maintaining the quality of cells in their pluripotent state, as well as improving aspects of experimental preparations, subpassaging and cryopreservation. Immediate targets for HESC protocol optimizations include improvements and standardization in growth and subpassaging conditions(1-4); feeder cell-free and ideally serum-free culture medium with each parameter optimized(5-10); maintenance of genomic and epigenomic stabilities; swiftly proliferating homogenous HESCs; reliable protocols for cell lineage-specific directed differentiation; and gene modification improvements(11). These procedures are intended for investigators experienced with sterile cell culture and, ideally, with mouse embryonic stem cell methodologies, just embarking on the thrilling and challenging new frontier of HESC research. The basic protocol describes the generation of mouse embryonic fibroblast (MEF) feeder cells to provide factors for the support and maintenance of HESCs in vitro. MEF feeder cells may be used to prepare conditioned medium (CM) to support a feeder-free culture 8, or HESCs can be grown directly on feeder cells. To confirm that HESC cultures remain in an undifferentiated state, it is important to confirm the presence of various pluripotency markers, included as options at the end of the protocol. Growth of hESCs in feeder-free conditions is optimal for studies examining genomics and transcriptomics, as it eliminates one source of contamination; however, feeder-free culture with CM does not preclude contamination of the cultures by zoonotic pathogens or by mouse proteins. Removal of these contaminants requires optimization either by complete elimination of feeder cells or by the use of human feeder cells.