Defects in the development of human germ cells are common, however, relatively little is known about the genes required for normal germ cell formation and function in men and women. Previously, we demonstrated the ability of human embryonic stem (ES) cells to differentiate into human germ cells in vitro and identified germ line specific genes involved in male and female germ cell development. The studies outlined in this proposal aim to enhance the formation of human germ cells from NIH-approved human ES cell lines by differentiating ES cells in the presence of Bone Morphogenetic Proteins (BMPs) and enriching for VASA-postive germ cells via FACS. Further characterization of human germ cell differentiation will be performed throughout development by examining the expression of germ cell specific genes at the mRNA level by quantitative RT-PCR and at the protein level by Western Blot analysis and immunohistochemistry. Functional analysis of germ cells differentiated from human ES cells will be accomplished by evaluating the expression of late germ cell markers, sex specific meiotic genes and meiotic spindle proteins, and by zona pellucida binding assays. In addition, the functional properties of both male and female human-ES cell derived germ cells will be assessed in vivo using a mouse transplantation system. Moreover, we also propose to silence the DAZ/DAZL genes, a family of candidate fertility factors, by RNA interference (RNAi) in human ES cells and determine the effects on male and female germ cell formation and function in vitro. Approximately 10-15% of couples in our population who are of reproductive age are infertile. This work will not only contribute to our understanding of the genetic requirements for normal human germ cell development, but it may also provide new therapeutic approaches for the diagnosis and treatment of infertility in men and women.
| Goyal, Akshi; Chavez, Shawn L; Reijo Pera, Renee A (2013) Generation of human induced pluripotent stem cells using epigenetic regulators reveals a germ cell-like identity in partially reprogrammed colonies. PLoS One 8:e82838 |
