National Centers for Translational Research in Reproduction and Infertility

Germ Cell Differentiation from Human iPSCs and hESCs

Stanford University

PI:  R.A. Reijo Pera

The fruit fly, Drosophila melanogaster, provides a powerful genetic system that combines natural and induced chromosomal deletions, transposon-mutagenesis, complementation and suppressor analysis. This has allowed elegant genetic dissection of gene function and interactions of diverse developmental pathways including those of germ cell development (see Project II for one example). In contrast, the genetic analysis of human germ cell formation and differentiation has historically been severely limited by genetic inaccessibility. Yet, the leading cause of infertility in both men and women is quantitative and qualitative defects in human germ cell (oocyte and sperm) development. Here, we seek to extend our previous studies on differentiation of human germ cells in order to incorporate the wealth of naturally-occurring human deletions and variants into a rigorous genetic dissection of the most common, molecularly-defined cause of failure in human germ cell development, deletion of the human AZFc {Azoospermia Factor-c) locus. We previously developed a system to differentiate human embryonic stem cells (hESCs) to germ cells. Then, we silenced and overexpressed genes that encode germ cell-specific cytoplasmic RNA-binding proteins (not transcription factors), and observed that we could modulate human male and female germ cell formation and developmental progression. We observed that human DAZL (Deleted in AZoospermia-Like) functions in female and male PGC formation and maintenance, whereas closely-related family members, BOULE and DAZ, promote entry into meiosis and development of haploid gametes with sperm-specific methylation patterns at imprinted loci in the male. We also conducted critical proof-of-concept studies in mice that showed that phenotypes observed in germ cell development in vitro mirrored phenotypes that were observed in vivo in wildtype, heterozygous, and Dazl-/- mutation-carrying mice and mouse ESCs (mESCs). Furthermore, transplantation of XX mESC derived oocytes resulted in recruitment of somatic cells to form follicles. These studies comprised the first direct experimental genetic analysis of human germ cell development and set the stage for extension to reprogrammed pluripotent stem cells (induced pluripotent stem cells (iPSCs)) in order to directly probe complex genetic variants linked to infertility. Our hypothesis is that human IPSCs will provide a rigorous genetic system, analogous to that of Drosophila, for functional dissection of human germ cell formation and common spontaneous deletions, mutations and variants. Further, we hypothesize that human germ cell development, from PGC formation and maintenance to meiotic entry and morphogenesis, is regulated by translational factors encoded by the human DAZ gene family in coordination with neighboring genes of the AZFc locus. We test these hypotheses with two specific aims: Aim 1. Compare germ cell differentiation from XX- and XY-bearing IPSCs to that of hESCs. Aim 2. Dissect the function of the most common genetic lesion implicated in human infertility via loss- and gain-of-function analysis of genes of the human AZFc region in IPSCs derived from men who present with Sertoli Cell Only (SCO) syndrome (completely lack a germ line).