Pluripotent stem cell (PSC) lines derived from embryonated avian eggs are a convenient platform for production of various recombinant proteins and vaccines. In chicks, both embryonic stem cells (ESC) and embryonic germ cells (EGC) are considered to be pluripotent cells obtained from early blastodermal cells (stage X) and gonadal tissues (stage HH28), respectively. However, the establishment and long-term maintenance of avian PSC lines faces several challenges and differs in efficiency between chick strains. This study aims to determine the effects of PSC culture media, including serum-based and serum-free media as well as various feeder layers, growth factors, and small molecules on derivation and maintenance of avian embryonic derived-PSCs. Our results have shown that among the different culture conditions, N2B27 serum-free medium supplemented with PD0325901 and SB431542, MEK and TGFβ chemical inhibitors, named as R2i and cytokine leukemia inhibitory factor (LIF) improved PSC derivation from stages X- and HH28 embryos. The application of N2B27/R2i + LIF medium validates the effect of defined pluripotency supporting medium on efficient derivation of chick PSCs and facilitates the use of these cells in biotechnology and biobanking of valuable species.

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