Objective: Among non-mammalian vertebrates, poultry, especially chicken SSCs are gaining remarkable importance in recent years, as biological models for basic research and also applications to produce transgenic poultry. There are only a few studies have focused on characterization and differentiation potential of the chicken SSCs in vitro. Identification of specific molecular biomarkers and differentiation capacity of chicken SSCs would not only help us to understand cell and molecular biology of these cells, but also can contribute to their applications in biotechnology. Thus, the present study was undertaken to search for reliable molecular markers specific to the one-day-old chicken SSCs and also we examined spermatogenesis potential of these cells in vitro by introduce a culturing condition to induce the spermatogenesis process in these cells. Methods: The testicular cells were isolated from one-day-old chickens testes by mechanical digestion. The presence of aligned cells, clusters and colonies and also alkaline phosphatase activity were investigated as preliminary criteria to detect SSCs in cell cultures. The molecular signature of SSCs was examined using RT- PCR analysis for DAZL, STRA-8, CVH, PLZF, SPRY-1, GFRα1, GDNF, POU5F1, NANOG, GPR125, THY-1, c-KIT, and BCL6B, and also by immunocytochemistry and flow-cytometry assays for POU5F1, GPR125 and SSEA-1. Furthermore, the spermatogenesis potential of one-day-old chicken SSCs was investigated by a 3D soft agar culture system. Results: The results of this study showed that colony-forming cells (SSCs) in one-day-old chicken testicular cell cultures were positive for alkaline phosphatase activity, and also expressed specific markers including DAZL, STRA-8, CVH, PLZF, SPRY-1, GFRα1, GDNF, POU5F1, NANOG, GPR125, THY-1, c-KIT, and BCL6B, at mRNA level. Moreover, these cells expressed POU5F1 and GPR125 proteins as reliable intracellular and cell surface markers, respectively; whereas they were negative for SSEA-1. Furthermore, we showed that one-day- old chicken colony-forming cells had spermatogenesis potential and thus could be produced sperm-like cells in a three-dimensional matrix in vitro. Conclusion: This study reports novel insights into the molecular signature of one-day-old chicken SSCs in comparison with mammalian SSCs and for the first time we report a successful protocol for in vitro spermatogenesis and thus production of sperm-like cells from one-day-old chicken testicular cell cultures.

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