Session 7, Abstract 41
IDENTIFYING THE EMERGENCE OF NASCENT GERMLINE CELLS FOLLOWING RHESUS MACAQUE IPSC DIFFERENTIATION IN VITRO
Ernesto J. Rojas* and Enrique Sosa (Amander T. Clark), UCLA, Dept. of Molecular, Cell, & Developmental Biology, 610 Charles E Young Dr. S. Los Angeles, CA 90095, USA
Currently there are no available options to restore fertility in pre-pubescent patients once lost to chemotherapy or radiation. A proposed technique is the induction of germline cells in vitro from induced pluripotent stem cells (iPSCs). In humans, the first stage of germline development involves the specification of primordial germ cells (PGCs) at the time of embryo implantation. In vitro PGCs are referred to as PGC-like cells (PGCLCs) to reflect their origin in the lab rather than in the embryo. The specification of PGCs/PGCLCs is required as a first step to make fertilization competent germline cells that could be used to restore reproduction. Our hypothesis is that the dynamics of PGCLC differentiation in vitro will reflect PGC formation in the embryo. Given the challenges of working in the peri-implantation period in human, we aim to perform these experiments using the rhesus macaque. We found that rhesus PGC markers are expressed in aggregates starting at day 1 of differentiation, but do not begin to colocalize until day 2. We also found it critical to develop triple immunofluorescence staining to simultaneously localize three different PGC genes relative to each other. The discovery that germ cell genes begin to colocalize in the same cell between 24-48 hours after aggregate induction, highlights the discovery that the first few hours of aggregate differentiation are critical to primate PGC specification. In future studies, we will look at different combinations of markers to elucidate which combination is best to identify the PGCLCs.