In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.

Avantea Laboratory of Reproductive Technologies Cremona 26100 Italy

Department of Genome Biology Graduate School of Medicine Osaka University Osaka 565 0871 Japan

Department of Stem Cell Biology and Medicine Graduate School of Medical Sciences Kyushu University Maidashi 3 1 1 Higashi ku Fukuoka 812 8582 Japan

Fondazione Avantea Cremona 26100 Italy

Freie Universitaet Berlin Berlin D 14195 Germany

Leibniz Institute for Zoo and Wildlife Research Berlin 10315 Germany

Technology Platform Pluripotent Stem Cells Max Delbrück Center for Molecular Medicine in the Helmholtz Association Berlin 13125 Germany

ZOO Dvůr Králové Dvůr Králové nad Labem 54401 Czech Republic

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In vitro fertilization program in white rhinoceros