Ectoderm derivatives have also been generated from hES cells and as with the mouse, most studies have focused on neuroectoderm and neural cells (Hornstein and Benvenisty 2004). The three CNS cell types can be derived from hES cells using several different protocols (Reubinoff et al. 2001; Zhang et al. 2001). When transplanted into newborn mice, hES-cell-derived neural cells generated neurons and glia that could be detected in different regions of the recipient brain 4 wk later (Zhang et al. 2001). Morphological analysis indicated that the donor cells had matured and were indistinguishable from the surrounding host cells. As observed with the mouse cells, coculture of hES cells with stromal cells led to the development of neural populations with midbrain dopamine characteristics (Perrier et al. 2004; Zeng et al. 2004). Cell populations displaying varying degrees of dopaminergic neuron differentiation have been transplanted into Parkinsonian rats (Ben-Hur et al. 2004; Zeng et al. 2004). Low numbers of human cells could be detected in the animals between 5 and 12 wk post-transplantation, and improvements in the behavior of some of the transplanted animals were noted in one of the studies (Ben-Hur et al. 2004). However, given that the graft that these animals received consisted of a mixture of neural and nonneural cells and that very few dopaminergic cells were detected in the recipients, the significance of these improvements is unclear. Future studies in which highly enriched populations of appropriate staged dopaminergic neurons are transplanted into a variety of different models will be required to determine the potential of this approach as a treatment of Parkinson's disease.
In addition to somatic tissues, several studies have documented the development of germ cells from differentiated ES cells. In the first study, ES cells were induced to differentiate in two-dimensional cultures in the presence of serum without additional factors (Hubner et al. 2003). To identify the developing germ cells in the differentiation cultures, the ES cells carried a modified Oct4 promoter that drove GFP expression specifically in the germline. GFP expression was detected as early as day 4 of differentiation, and by day 8 up to 40% of the population was positive. By day 26 of culture, oocyte-like cells enclosed in a structure resembling a zona pellucida were released into the culture from the differentiating cell mass. Following several additional weeks of culture, structures similar to blastocysts were observed, likely originating from the parthenogenic activation of the oocytes.
Male germ cell development has also been demonstrated in ES cell differentiation cultures. With one approach, developing germ cells were identified using an ES cell line in which GFP was targeted to the mouse vasa homolog (Mvh), a gene specifically expressed in germline cells (Toyooka et al. 2003). While GFP+ cells were detected in EBs stimulated with serum alone, the number was significantly enhanced when the EBs were coaggregated with a BMP4-expressing cell line. This observation is encouraging as BMP4 is a known regulator of germ cell development in the mouse embryo (Lawson et al. 1999). To determine the potential of these putative germ cells, GFP+ cells were isolated from the EBs, cocultured with gonadal cells, and then transplanted under a host testis capsule, where they were shown to participate in spermatogenesis. In the second study, germ cells generated in serum-stimulated EBs were isolated on the basis of SSEA1 expression and cultured in the presence of retinoic acid, LIF, bFGF, and c-kit ligand (Geijsen et al. 2004). Cells differentiated under these conditions displayed imprint erasure, a distinguishing characteristic of germ cells. If maintained in culture for 2 wk, these serum-stimulated EBs generated a small population of spermatid-like cells that could be isolated using an antibody recognizing the sperm acrosome. This population was enriched for haploid cells that were able to complete fertilization, following injection into oocytes.
Germ cell development has also been analyzed in hES cell differentiation cultures (Clark et al. 2004). In this study, gene expression profiles suggested the development of both male and female germ cells in serum-stimulated EBs. However, as the cells were not isolated and further characterized, the efficiency of differentiation and the extent of lineage maturation are unknown.
The findings from these different studies clearly demonstrate that it is possible to generate germ cells from mouse ES cells and, in doing so, open new and exciting avenues for researchers to study the development of this lineage. Before the model can be widely used for such studies, however, it will be important to define the regulators that induce germ cell development, preferably in the absence of serum. With appropriate conditions, it should be possible to reproducibly generate large numbers of such cells.
The studies reviewed in the previous sections indicate that it is possible to generate derivatives of the three primary germ layers as well as germ cells from ES cells differentiated in culture. They also highlight the fact that the efficiency in forming different cell types varies considerably as neuroectoderm and certain mesoderm derivatives including the hematopoietic, vascular, and cardiac lineages are reasonably easy to generate, whereas the development of definitive endoderm and its derivatives such as mature hepatocytes and pancreatic -cells are considerably more challenging. Given the complexity of lineage development in the early embryo, it is remarkable that it is possible to generate any mature populations in a reproducible manner from differentiated ES cells. While the successes to date highlight the importance and potential of this model system, significant improvements in lineage development, in particular endoderm and its derivative populations, will ultimately depend on gaining a better understanding of the events regulating the induction of the primary germ layers. To achieve this, it will be important to recapitulate the developmental events of the early embryo in the ES cell system, to use serum-free conditions and defined molecules for differentiation, and to develop reporter systems that enable one to quantify lineage development.