Making Germ Cells from Human Embryonic Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 77

3.6 The Future: Improved Methods

and Clinical Implications

In future studies it will be necessary to carefully dissect and characterize
in vitro -derived germ cells at the single-cell level. Careful examinations and com-
parisons can then be made with single ES-derived gametes and mature human
sperm and eggs. Using advanced microarray technologies for the analysis of
genetic variants and cellular gene expression will be highly useful in dissecting the
protein and genetic make-up of ES-derived germ cells. In addition, improved imag-
ing techniques might also allow stringent comparative analysis between the mor-
phology and ultrastructure of germ cells and gametes derived both in vivo and
in vitro . Most importantly, careful analysis of successful meiosis, genomic methyla-
tion, chromatin status, nuclear architecture, and chromosome ploidy of ES cell-
derived sperm and eggs will be paramount to assess quality and functionality,
especially with respect to human gametes.

3.6.1 Improving Culture Conditions for Enhanced

Gamete Formation

The process of germ cell formation, differentiation, and maturation in the fetal and
adult gonad consists of a series of complex molecular and cellular events. The devel-
oping germ cells and gametes are in constant contact with the somatic support cells
and hormone/secretory cells of the gonad. In fact, the close physical connections and
intercellular signaling are apparent in both sperm with Sertoli cells, and oocytes with
granulosa and theca cells of the follicle. Therefore, it is logical to speculate that the
generation of more mature gametes from ES cells in culture will require certain key
elements of the somatic gonadal niche. Recapitulating the in vivo factors and cellular
environment will likely be crucial for the development and further maturation of
germ cells in vitro . Indeed, the most successful investigations have used co-culture
gonadal cell systems, gonadal transplantation strategies, and/or signaling factors
known to exist within the gonad (Fig. 3.7a ). One recent study used the co-culturing
of human fetal gonadal stromal cells with human embryonic or induced pluripotent
stem cells to greatly enhance the formation of germ cells (Park et al. 2009 ). Using
ovarian and testicular cells/aggregates or using cell lines developed from fetal
gonadal somatic cells may provide an effective system to enhance the development
and maturation of normal functional human gametes in vitro (Fig 3.7b ).

3.6.2 Clinical Implications and Outlook

Development of functional mature gametes is a key event for sexually reproducing
species. Establishment of an in vitro cell-based system, like that of human ES cells,