The molecular signature and spermatogenesis potential of newborn chicken spermatogonial stem cells in vitro Sajjad Sisakhtnezhad & Ahmad Reza Bahrami & Maryam M. Matin & Hesam Dehghani & Madjid Momeni-Moghaddam & Sohrab Boozarpour & Moein Farshchian & Mahtab Dastpak Received: 28 July 2014 /Accepted: 28 October 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014 Abstract Although chicken spermatogonial stem cells (SSCs) have received considerable attention in recent years, only a few studies so far have focused on their derivation and characterization 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 applica- tions in biotechnology. In this regard, we found that colony- forming cells (SSCs) in newborn chicken testicular cell cul- tures 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 pro- teins as reliable intracellular and cell surface markers, respec- tively; whereas they were negative for SSEA-1. Furthermore, we showed that newborn chicken colony-forming cells had spermatogenesis potential and thus could be produced sperm- like cells in a three-dimensional matrix in vitro. In conclusion, this study reports novel insights into the molecular signature of newborn 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 newborn chicken testicular cell cultures. Keywords Gallus gallus . Spermatogonial stem cell . Molecular markers . In vitro spermatogenesis . 3D matrix Introduction Spermatogonial stem cells (SSCs) are the main source of cells for spermatogenesis which persists in the testis (Zohni et al. 2012). They are valuable cells with applications in develop- mental biology, transgenesis technology, and clinic (Mizrak et al. 2010; Vlajkovic et al. 2012). In this regard, the poultry SSCs have served as a biological model for medical research and also for production of recombinant proteins. The latter has been achieved by manipulating these cells in vitro, using non- viral-based vectors and re-injecting them into rooster testis, as the most efficient and cost effective strategy to produce trans- genic poultry, which offer a powerful bioreactor for the pro- duction of recombinant proteins in eggs (Li and Lu 2010; Yu et al. 2010). Progress in this field is dependent on efficient and robust techniques for isolation and identification of the SSCs in chickens and other organisms. S. Sisakhtnezhad : A. R. Bahrami : M. M. Matin : M. Farshchian : M. Dastpak Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran S. Sisakhtnezhad : A. R. Bahrami (*) : M. M. Matin : H. Dehghani : M. Farshchian : M. Dastpak Cell and Molecular Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, P.O. Box: 9177948974, Iran e-mail: Ar-Bahrami@um.ac.ir H. Dehghani Department of Basic Science, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran M. Momeni-Moghaddam Department of Biology, Faculty of Science, Hakim Sabzevari University, Sabzevar, Iran S. Boozarpour Department of Biology, Faculty of Basic Science, Gonbad Kavous University, Gonbad Kavous, Iran Present Address: S. Sisakhtnezhad Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-014-9843-1