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 chromatography 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 activators of transcription-3 by LIF, which is sufficient to maintain the undifferentiated state of ES cells, was detected by Western blotting analysis.
Horiuchi
Hiroyuki
H
Laboratory of Immunobiology, Department of Molecular and Applied Biosciences, Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima, Japan.
Furusawa
Shuichi
S
Matsuda
Haruo
H
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002642:Chick Embryo / Q000166:cytology* / Q000378:metabolism; D002645:Chickens / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics / Q000494:pharmacology; D053605:Leukemia Inhibitory Factor; D016256:Lewis X Antigen / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D050796:STAT3 Transcription Factor / Q000378:metabolism; D014170:Transformation, Genetic
D018076:DNA, Complementary; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D016256:Lewis X Antigen; C507838:Lif protein, mouse; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; D000469:Alkaline Phosphatase