Byline: Arazdordi Toumadje (1), Ken-Ichi Kusumoto (1), Angela Parton (2), Patricia Mericko (1), Lori Dowell (2), Guozhong Ma (1), Luping Chen (1), David W. Barnes (2), J. Denry Sato (2) Keywords: ES cell; embryoid body; immunocytochemistry; flow cytometry Abstract: Although the ES-D3 murine embryonic stem cell line was one of the first derived, little information exists on the in vitro differentiation potential of these cells. We have used immunocytochemical and flow cytometric methods to monitor ES-D3 embryoid body differentiation in vitro during a 21-d period. Spontaneous differentiation of embryoid body cells was induced by leukemia inhibitory factor withdrawal in the absence of feeder cells. The pluripotent stem cell markers Oct-3/4, SSEA-1, and EMA-1 were found to persist for at least 7 d, whereas the primitive endoderm marker cytokeratin endo-A was expressed at increasing levels from day 6. The localization of these antigens within the embryoid bodies suggested that embryonic ectoderm- and primitive endoderm-derived tissues were segregated. Localized expression of class III beta-tubulin and sarcomeric myosin also was detected, indicating that representatives of all three embryonic germ layers were present after induction of differentiation in vitro. Author Affiliation: (1) National Stem Cell Resource, American Type Culture Collection, 10801 University Boulevard, 20110, Manassas, Virginia (2) Mount Desert Island Biological Laboratory, Old Bar Harbor Road, P.O. Box 35, 04672, Salisbury Cove, Maine Article History: Registration Date: 22/03/2003 Received Date: 11/09/2003 Accepted Date: 29/12/2003 Article note: These authors contributed equally to this study.