Detection of Embryonic Gonadal Stem Cells Following Short-Term Culture Using Flow Cytometry and Cell-Transfer.

The incorporation of artificial reproductive technologies, including stem cell rescue, could enhance the reproductive success of an avian breeding/management program. The goal of this research is to produce host chickens capable of generating donor-derived sperm. This could be accomplished by transfer of donor embryo germ stem cells (eGSCs) from exotic birds to chicken host embryos during migration of the endogenous primordial germ cells at stages 15–17 (2.5 days of incubation). The objective of this study is to determine the feasibility of culturing eGSCs isolated from embryonic gonads for three days, the time needed for a host embryo to reach stages 15–17, when it will be ready to receive donor eGSCs. Mid-incubation (stage 37–39) chicken embryo gonads were digested to single-cell suspension. The percent of eGSCs present in the fresh, uncultured (0d) and following a three-day (3d) culture in stem cell medium was measured by flow cytometry and stem cell specific antibodies (SSEA-1, SSEA-3, SSEA-4, CD9, integrin a6, and OCT3/4). To determine the ability of the cultured eGSCs to colonize the host gonad, both 0d and 3d cells were stained with PKH26, transferred to host chicken embryos, incubated for an additional 7 days, and quantified by flow cytometry. EGSCs expressing SSEA-1 (0d: males (M):14.4%; females (F): 2.6%; 3d: M: 14.4%; F: 16.5%); SSEA-3 (0d: M: 4.2%; F: 3.2%; 3d: M: 44.9%; F: 38.8%); SSEA-4 (0d: M: 1.7%; F: 2.3%; 3d: M: 96.0%; F: 94.6%); and CD9 (0d: M: 0.7%; F: 1.6%; 3d: M: 3.2%; F: 7.5%) increased in numbers from 0d to 3d culture. However, the cells expressing integrin-a6 (0d: M:6.2%; F: 5.0%; 3d: M: 2.3%; F: 1.4%) and OCT3/4 (0d: M: 14.8%; F: 15.9%; 3d: M: 7.2%; F: 7.3%) decreased. The percent of host gonads that contained uncultured (od) donor cells was 43% and 80% for male-to-male and female-to-female transfers, respectively. In contrast, the percent of positive gonads were 40% and 20% for male-to-male and female-to-female transfers, when donor cells were cultured for 3d. This study demonstrates that culturing embryonic gonad cell suspensions for three days is a practical method of maintaining cells until incubated host embryos reach stage 15–17.