CSIRO PUBLISHING Reproduction, Fertility and Development, 2009, 21, 696–708 www.publish.csiro.au/journals/rfd Multipotential ability of primitive germ cells from neonatal pig testis cultured in vitro Sandeep Goel A,C , Mayako Fujihara A , Kazuo Tsuchiya B , Yuji Takagi B , Naojiro Minami A , Masayasu Yamada A and Hiroshi Imai A,D A Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. B Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan. C Centre for Cellular and Molecular Biology, Council for Scientific and Industrial Research, Uppal Road, Hyderabad 500 007, India. D Corresponding author. Email: imai@kais.kyoto-u.ac.jp Abstract. Gonocytes are progenitor-type germ cells that arise from primordial germ cells and differentiate further into spermatogonia, thereby initiating spermatogenesis. In the present study, freshly isolated gonocytes were found to have either weak or no expression of pluripotency determining transcription factors, such as POU5F1, SOX2 and C-MYC. Interestingly, the expression of these transcription factors, as well as other vital transcription factors, such as NANOG, KLF4 and DAZL, were markedly upregulated in cultured cells. Cells in primary cultures expressed specific germ cell and pluripotency markers, such as lectin Dolichos biflorus agglutinin (DBA), KIT, ZBTB16, stage-specific embryonic antigen (SSEA-1), NANOG and POU5F1. Using a monoclonal antibody to specifically identify porcine germ cells, the stem cell potential of fresh and cultured cells was determined with a testis xenotransplantation assay. Colonised porcine germ cells were detected only in mouse testes that were either transplanted with fresh testicular cells or with cells from primary cultures. Interestingly, testes transplanted with cells from primary cultures showed colonisation of germ cells in the interstitial space, reflecting their tumourigenic nature. The formation of teratomas with tissues originating from the three germinal layers following the subcutaneous injection of cells into nude mice from primary cultures confirmed their multipotency. The results of the present study may provide useful information for the establishment of multipotent germ stem cell lines from neonatal pig testis. Introduction The testes are a source of germline stem cells that are capa- ble of transmitting genetic information to the next generation. These cells have emerged as an alternative to embryonic stem (ES) cells for targeted genetic modification in mice (Kanatsu- Shinohara et al. 2006). Because there is no germline-competent ES cell line in large domestic species at present, male germline stem cells could be used for the production of animals with targeted mutations using the homologous recombination tech- nique. However, in order to for this to be achieved, a long-term culture system needs to be developed for these cells. Although a long-term culture system for spermatogonial stem cells has been developed for the mouse (Nagano et al. 1998, 2003; Kanatsu- Shinohara et al. 2003, 2005; Kubota et al. 2004; Guan et al. 2006), rat (Hamra et al. 2005; Ryu et al. 2005) and hamster (Kanatsu-Shinohara et al. 2008a), such a system is not available for any large domestic species. Gonocytes are primitive germ cells that exist in neona- tal testes. Recently, we identified specific markers for porcine gonocytes and used these markers to identify gonocytes during purification and short-term culture in vitro (Goel et al. 2007). Although we were able to isolate gonocytes and culture them in vitro for 1 week, we were unable to determine whether they could be passaged and whether they had stem cell potential. In the same study, we examined the expression of crucial pluri- potency determining factors, such as NANOG and POUF51, in developing postnatal pig testes (Goel et al. 2007). How- ever, the expression of these and other essential markers has not been defined in porcine cultured male germ cells. Although pig spermatogonia cultured in vitro have been characterised for the expression of some markers, such as POU5F1, RET, UCHL-1 and KIT (Luo et al. 2006), the stem cell potential of these cultured spermatogonia remains unknown. Stem cells in a testicular germ cell population can be identified using the testis transplantation assay (Brinster and Zimmermann 1994). This technique provides a functional assay for male germ stem cells, which are the only cells that can colonise recipient mouse testes. Previous studies have shown that germ cells from xenogenic hosts, such as rabbit and dog (Dobrinski et al. 1999), as well as from large domestic species, such as pig, cattle and horse (Dobrinski et al. 2000), can colonise recipient mouse seminiferous tubules. © CSIRO 2009 10.1071/RD08176 1031-3613/09/050696