Maintenance of Chicken Embryonic Stem Cells In Vitro

In this chapter, we describe the methods we have used to show that chicken leukemia inhibitory factor (LIF) maintains chicken embryonic stem (ES) cells in an undifferentiated state in culture. Recombinant chicken LIF (rchLIF) was expressed as a fusion protein linked to glutathione S-transferase (GST) and purified to greater than 90% purity in two chro-matography stages, the first an affinity step using the GST tail, which was cleaved before further purification by gel chromatography. Chicken ES cells were obtained by culturing chicken blastodermal cells isolated from stage X embryos of freshly laid chicken eggs. These cells can be maintained in media containing rchLIF for at least 9 d without any other cytokines or feeder cells. Chicken ES cells were characterized by the expression of alkaline phosphatase activity, stage-specific embryonic antigen (SSEA)-1 and embryonal carcinoma cell monoclonal antibody-1. In addition, the phosphorylation of signal transducers and acti-vators of transcription-3 by LIF, which is sufficient to maintain the undifferentiated state of ES cells, was detected by Western blotting analysis.