Effective Derivation and Manipulation of Mouse Embryonic Stem Cells

Abstract

Mouse embryonic stem (ES) cells were first isolated in 1981 from the inner cell mass of the blastocyst before implantation within the uterine wall. ES cells have the unique property of pluripotency and also of capable of infinite self-renewal. They can be maintained in undifferentiated state in culture or be differentiated to multilineage cell types from all three embryonic layers, both in vivo and in vitro (Evans & Kaufman, 1981; Martin, 1981; Brook & Gardner 1997). These properties of ES cells have made them an extremely interesting and important tool for basic and applied research, especially for the studies of embryogenesis, gene function, and development. However, current protocols for mouse ES cell derivation are often very inefficient and require a great deal of specialized training and expertise, the potential of mouse ES cells has not yet been fully and systematically exploited. There are numerous protocols available for mouse ES cell line derivation from blastocysts, and interestingly the effectiveness of derivation of ES cells is largely based on the mouse strain. In practice the efficiency of derivation in strains other than 129 does not usually exceed 10% (Bryja et al., 2006; Batlle-Morera et al., 2008). Moreover, some protocols require the use of sophisticated techniques, such as isolation of inner cell mass via immunosurgery of intact blastocysts, isolation of epiblast cells from implanted the egg cylinderstage embryos, or selective ablation of differentiated cells. Other variations could include derivation on feeder layers, the absence of supporting feeders all together, or the use of conditioned media or the use of serum replacement (McWhir et al., 1996; Schoonjans et al., 2003; Cheng et al., 2004; Tesar, 2005; Bryja et al., 2006; Doungpunta, et al., 2009). Rho kinase inhibitor Y-27632 and the dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging (Watanabe, et al., 2007; Ruchi, et al., 2008), and since then we have adapted these methods in our mouse ES cell derivation protocol. Our data demonstrates that Y-27632 and Accutase increase the efficiency of mouse ES cell derivation, and the resultant ES cells retain developmental pluripotency (including stable karyotype, surface markers, teratoma formation, and the ability to undergo germline transmission). In this chapter, we describe a simple and efficient protocol for derivation of mouse ES cells and provide details on how to culture and manipulate the resultant cells. As compared to other available protocols, this method does not require special equipment, genetic modification, or advanced training other than regular tissue culture and animal handling skills. It is our hope that this protocol will allow investigators new to the ES field