Chapter 9 Genetic Approaches in Human Embryonic Stem Cells and their Derivatives: Prospects for Regenerative Medicine

This chapter reviews current protocols to maintain human embryonic stem cells (hESCs), genetic approaches to modifying undifferentiated hESCs, differentiation of hESCs into multiple lineages and transplantation of their derivatives, and genetic manipulation of hESC-derived progenies and also discusses the potential applications of genetic modifications of hESCs and their derivatives in the context of regenerative medicine. Knock-in/knockout technologies based on homologous recombination are the most widely used methods to study gene function in most mammals. Homologous recombination in hESCs is important for modifying specific hESC-derived tissues for therapeutic applications in transplantation medicine. In vitro studies of hESCs involved in understanding the pathogenesis of gene disorder diseases such as Wiskott-Aldrich syndrome or cancer also need the loss-and-gain methods. Synthetic chemicals such as cationic lipids are extensively used for the delivery of DNA into hESCs. These chemical approaches are based on the neutralization of cationic lipids to negatively charged DNA followed by the formation of DNA/lipid complexes, which possess an excess of positive charges. These complexes bind to the negatively charged membranes of hESCs and are subsequently taken by the cells through endocytosis. Retroviral vectors are used for stable gene transfer into mammalian cells. They are derived from retroviruses. This family consists of seven genera that include alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus, and spumavirus.