The studies reviewed in the previous sections highlight the successes to date in differentiating cell populations from ES cells and in demonstrating the potential of using the model to investigate early development and generating cells for replacement therapy. As progress is made in establishing defined culture systems and selection strategies for the generation of highly enriched lineage-specific populations, the ES/EB model will be used in many different areas of biology and medicine. The following section and the summary in Figure 7 highlight three of these areas.
Developmental biology The ability to access and manipulate populations representing early developmental stages in the ES cell differentiation cultures provides a new approach for addressing questions of lineage commitment. This system provides a model of early mammalian development that enables manipulations comparable to those carried out in other organisms such as
Xenopus and zebrafish. As outlined in the previous sections, the ES cell system has already been used to study the role of specific genes in development; to isolate early-stage populations; to identify progenitors that represent novel stages of hematopoietic, vascular, and neural development; and to begin to define the factors that regulate primary germ layer induction.
A critical area of future investigation is the regulation of germ layer induction and tissue specification using the ES cell differentiation cultures. These issues are most easily addressed using lineage-specific markers that allow quantification of the response to the specific factor or sets of factors being tested. When appropriate cell surface markers are not available, selectable markers can be targeted to lineage-specific genes as demonstrated for brachyury (Fehling et al. 2003
), Pdx1 (Micallef et al. 2005), and Sox1 (Ying et al. 2003b). Given their role in the development of the early embryo, factors from the TGF
, Wnt, and FGF families would be obvious candidates to be tested. The outcome of these experiments will establish a role for the different factors in these early developmental steps and ultimately lead to the establishment of defined conditions that will enable efficient and reproducible lineage-specific induction in both mouse and human ES cell differentiation cultures.
The ES cell model is well suited for the identification, isolation, and characterization of cell populations representing the early stages of lineage commitment. The identification of EPL cells (Rathjen et al. 1999) and the isolation of mesoderm and neuroectoederm, through targeting of GFP to brachyury (Fehling et al. 2003) and Sox1 (Ying et al. 2003b), respectively, highlight the advantages of this model in isolating cell populations that are difficult to access in the early embryo. It will be important to expand this approach to introduce different selectable markers into genes that define distinct developmental steps within a given lineage. These strategies will not only provide progenitors for cell-based therapy, they will also generate closely related populations that can be used for the identification of genes involved in the development and maturation of the lineage under investigation.
Once genes have been identified, the ES cell system is an ideal model with which to study their role in lineage development. Gain-of-function studies can now be carried out with inducible systems that allow the expression of the gene of interest at specific stages in specific cell populations (Kyba et al. 2002). The ability to regulate expression through inducible vectors is an important consideration as certain genes display different functions in different cell types. Loss of function can be routinely achieved though homologous recombination (Joyner 1991). For in vitro analysis, both alleles of the gene under study need to be disrupted to create a homozygous null ES cell line. Although this approach can and has been used to study the role of different genes in early lineage commitment, disruption of function though homologous recombination is slow and labor-intensive and thus not appropriate for the analysis of large numbers of genes. An alternative promising strategy for down-regulating gene expression is through the use of RNAi (Dykxhoorn et al. 2003) that can be delivered to both mouse and human ES cells with lentiviruses (Hamaguchi et al. 2000; Ma et al. 2003). Several studies have demonstrated that RNAi can be used to modify gene expression in undifferentiated ES cells as well as in EBs (Velkey and O'Shea 2003; Matin et al. 2004; Zippo et al. 2004). If future work shows that this approach is effective for a broad range of genes at different developmental stages, the use of RNAi will become a routine technology for gene function analysis in ES cell differentiation cultures.
All of these strategies can be applied to the human ES cell system. Of particular importance will be the investigation of the early developmental steps and the analysis of gene function, as these issues obviously cannot be addressed in the human embryo. With respect to loss-of-function approaches, there is only one report to date of targeting through homologous recombination (Zwaka and Thomson 2003). Whether or not gene targeting in hES cells will become a "routine" technology as it is with mouse cells remains to be determined. An important avenue of investigation with enormous potential is the generation and analysis of hES cell lines with genotypes characteristic of different diseases. Such lines have already been established from embryos carrying genetic diseases, identified through preimplantation diagnosis (Verlinsky et al. 2005). hES cell lines from patients suffering from a variety of different diseases can be generated through the use of somatic cell nuclear transfer (SCNT) (Hochedlinger and Jaenisch 2003). Such ES cell lines could be used to study the effects of specific disease mutations on lineage development, to establish new strategies to investigate the underlying causes of certain diseases, and as a model for testing new approaches for treating the diseases.
Answers to all your Biology Questions
