Maintenance, Propagation, and Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Abstract

The methods described here for maintenance, propagation and freezing of human embryonic stem cells (hESCs) represent a combination of protocols obtained from different sources, and have been extensively tested and are routinely used to culture different hESC lines. As it has been demonstrated that hESCs and induced pluripotent stem cells (iPSCs) are very similar to one another, the same protocols are used for both cell types. In addition to standard maintenance techniques, we describe a method in this chapter to assess the pluripotency of hESCs through the formation of embryoid bodies (EBs). As is the case for culture techniques, several protocols for EB formation exist and certain EB protocols work better for Hues hESC lines than for H hESC lines, and vice versa. Although many of the methods for hESC culture are similar to standard cell culture techniques, greater care and vigilance need to be exercised to maintain the phenotypic and genotypic properties of hESCs. In particular, many established nonprimary cell lines frequently harbor bacterial contaminants, such as mycoplasma, and are able to thrive while maintained in antibiotic-containing media. In contrast, mycoplasma-contaminated hESCs grow poorly, fail to form EBs, and will eventually die. Owing to their sensitivity to any level of contamination, we highly recommended establishing a culture facility dedicated to hESC work only. In this chapter, we outline detailed procedures on how to grow and propagate hESCs (and iPSCs), include detailed protocols for the characterization of hESCs, and provide specific methods for the in vitro differentiation of hESCs via EB formation.