229 DERIVATION OF PRESUMPTIVE GONOCYTES IN VITRO FROM PRIMATE EMBRYONIC STEM CELLS

Abstract

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis, because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES (cyES) cell lines and attempted to induce their differentiation into germ cells in order to obtain further information on the development of primate germ cells by observing the transcripts of some markers reported as specific for germ cells. CyES cell lines were established using blastocysts produced by intracytoplasmic sperm injection (ICSI). For inducing superovulation, females were treated with 25 IU kg^-1 pregnant mare serum gonadotropin once a day for 9 days, followed by 400 IU kg^-1 hCG. Oocytes were collected at 40 h after injection of hCG. After sperm injection, embryos were cultured in mCMRL medium to the blastocyst stage. For cyES cell establishment, inner cell masses (ICMs) were isolated by immunosurgery. The ESC colonies developed at about 10 days after ICM plating, and 3 cell lines were successfully established (3/11; 27.3%). All cell lines expressed Oct3/4, SSEA-4, and ALP activity. These ESCs formed teratomas containing 3 different embryonic layers when injected into SCID mice. And the cells could be passaged over 50 times without losing their original properties. To observe in vitro gametogenesis, we attempted to induce differentiation by non-adherent conditions. When cyES cells differentiated spontaneously, the aggregated structures (i.e. embryoid bodies; EBs) accumulated vasa, the expression of which is restricted to germ cells, and some meiotic markers such as dmc1 and sycp1 that exist only in synaptonemal complexes in meiosis. The existence of these markers was also confirmed by immunocytochemistry on cryosections. Interestingly, these products expressed oct4 and nanog again at Day 16, though the expression of both genes diminished at once with onset of differentiation. In vivo, it is reported that vasa, oct4, and nanog are expressed in migrating PGCs, posibly throughout the development of germ cells into spermatocytes/oocytes. Given the results obtained with the meiotic markers, it is possible that developing germ cells such as PGCs or gonocytes could be formed in cynomolgus EBs as in previous cases with mouse or human EBs. These results demonstrate that cyES cells might contribute to putative germ cells in vitro by differentiating into EBs and could be used as a model for studying mechanisms of germ cell development.

This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.