Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture

Abstract

The use of a transgenic line of rats that express enhanced GFP (EGFP) exclusively in the germ line has allowed a separation of feeder layers and contaminating testis somatic cells from germ cells and the identification of a set of spermatogonial stem cell marker transcripts. With these molecular markers as a guide, we have now devised culture conditions where rat spermatogonial stem cells renew and proliferate in culture with a doubling time between 3 and 4 days. The marker transcripts increase in relative abundance as a function of time in culture, and the stem cells retain competency to colonize and develop into spermatids after transplantation to the testes of recipient rats. The cells also remain euploid after at least 12 passages. Cell lines could be isolated and cryopreserved and, upon subsequent thawing, continue to self renew. Transfection of the spermatogonial stem cells with a plasmid containing the neomycin phosphotransferase (neo) selectable marker resulted in selection of G418-resistant cell lines that effectively colonize recipient testes, suggesting that gene targeting is now feasible in the rat.germ line ͉ spermatogenesis ͉ gene targeting ͉ enhanced GFP fluorescence T he laboratory rat represents one of the most comprehensively studied mammalian species, with described use in Ͼ1 million publications in a wide range of medically relevant areas. Qualities such as size, fecundity, behavior, ease of surgical techniques, tissue sampling, and general laboratory management have contributed to its popularity (1-3). However, a failure to develop technology to produce rat genetic models through gene targeting has resulted in the mouse becoming a widely popular animal model.Although mouse embryonic stem (ES) cells renew with a sense of immortality, primitive hemapoietic stem cells self renew ineffectively and for only a short period in vitro (4, 5). ES cells from species other than the mouse or human fail to generally self renew effectively and also lose pluripotency in culture (6). We and others have not succeeded in culturing pluripotent ES cells from the rat; however, if spermatogonial stem cells could be cultured under conditions where they self renewed and expanded in numbers, conceivably they could also be genetically modified in vitro in much the same manner as seen with mouse ES cells. This alternative to the use of genetically modified ES cells would result in direct germ line transfer and an escape from the intervening formation of a mosaic animal. After appropriate selection of gene-targeted cells in culture, the chosen spermatogonial stem cells could either be induced to differentiate to the haploid stage in vitro or transplanted to the testes of recipient rats to allow development to the haploid stage. In either case, intracytoplasmic sperm injection into the egg would result in transmission of the genetically modified information (7,8).We now show that genetically marked spermatogonial stem cells from transgenic germ-cell-specific EGFP (GCS-EGFP) rats (9) can be placed under defined culture co...

Ectopic porcine spermatogenesis in murine subcutis: tissue grafting versus cell-injection methods

“…These GS cells can expand in vitro [16]. Manipulation of their genome has also become possible [27,28]. To extend this technique to other mammals such as livestock and primates, an experimental system to induce spermatogenesis from GS cells is necessary.…”

Can we grow sperm? A translational perspective on the current animal and human spermatogenesis models

“…7,8 The functional characteristics of the SSC and their interaction with the seminiferous tubule and immune cells have also been further explored. [10][11][12][13] Furthermore, by modifying the SSC prior to transplantation, transgenic animals can be generated using this technique. [14][15][16][17] Clinically, there may be potential to apply the SSC transplantation model in humans, especially in the fertility preservation of treated pre-pubertal male cancer patients (as outlined in Figure 1 18 ).…”

Brief History, Pitfalls, and Prospects of Mammalian Spermatogonial Stem Cell Research

“…In contrast, the culture of SSCs is only now being developed. The culture conditions for rat and hamster GS cells have been established (Hamra et al 2005;Ryu et al 2005;Kanatsu-Shinohara et al 2008a). Although these results suggest a promising future for the manipulation of SSCs, their application remains limited because of factors such as low fertility (Brinster and Avarbock 1994;Ogawa et al 1999a).…”