Genetic Modification of Human Embryonic Stem Cells (2007)

Embryonic stem (ES) cells, originated from the inner cell mass (ICM) of a blastocyst embryo, are self-renewable (Evans and Kaufman, 1981; Martin, 1981). The pluoripotent nature of ES cells endows them as a great tool for dissecting cell lineage development in mammals and they can be used as a limitless source for producing specialized cells for potential cell therapy. Application of gene targeting technology in mouse ES cells has allowed the cloning of mice with modified genomes (Frohman and Martin, 1989; Koller et al., 1989), thus significantly widening the use of mice as a vertebrate experimental model in biological research today. Forced overexpression or knockout of critical genes by genetic modification has become a routine technique in mouse ES cells to create transgenic mice for analyzing gene function and genetic pathways in the context of intact animals and in cell lines derived from such animals. Gene targeting in mouse ES cells also allows the creation of mouse models of human diseases (Bedell et al., 1997a; Bedell et al., 1997b), which offers insights into the genetic, biochemical and pathological basis of the diseases and may help developing treatments.

Like their murine counterparts, human ES cells (Reubinoff et al., 2000; Thomson et al., 1998) offer invaluable and perhaps the only system for directly studying early human development as manipulation of human embryos is prohibited and information gained from model organisms (Anderson and Ingham, 2003) does not always reflect what is occurring in humans. The ability of human ES cells to produce almost any cell types in our body brings hopes to many incurable diseases affecting the central nervous system (CNS) (Dunnett et al., 2001; Silani et al., 2004), pancreas (Bonner-Weir and Weir, 2005), and heart (Srivastava and Ivey, 2006), etc. The potential use of human ES cells can be further enhanced by genetic modification (Drukker, 2005; Kobayashi et al., 2005). Application of currently available genetic modification techniques in human ES cells will provide researchers with even more versatile tools to study early human development and to expand the potential use in medicine. Basic mechanisms underlying early human development can be dissected through interfering with specific signaling pathways by forced gene overexperssion or silencing. Genetic defects may be corrected in human ES cells by site directed gene modifications, which may lead to the development of treatment for many inheritable diseases (Chang et al., 2006; Rideout et al., 2002). By labeling cells with tissue specific markers, genetic modification may help us obtain pure functional cells of desired types after differentiation, which can be used as more effective transplant medicine.

Unlike their murine counterparts, genetic modification in human ES cells has been technically challenging. Standard chemical or mechanical methods for gene delivery exhibit very low efficiency in human ES cells, with the most effective approaches yielding only approximately one stable transfectant per 105 transfected cells (Eiges et al., 2001; Zwaka and Thomson, 2003). After transfection, transgene expression appears to be suppressed more severely in human ES cells than in mouse ES cells (Xia, 2006). Thus successful examples are still rare. In this review, we will summarize the current methodologies for genetic modification of human ES cells from very limited literatures and propose future directions to overcome the present problems in genetic manipulation of human ES cells.