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a Institut für Anatomie, Lehrstuhl für Anatomie und Entwicklungsbiologie, Universitätsklinikum Essen, Essen, Germany;
b Zentrum Anatomie, Abteilung Anatomie/Embryologie, Universität Göttingen, Göttingen, Germany;
c Stiftung caesar, Bonn, Germany
Key Words. Embryonic stem cell • Rhesus monkey • Primate • Differentiation • Epithelial–mesenchymal transition • Gastrulation • Brachyury • Snail2 • E-cadherin
Correspondence: Hans-Werner Denker, Prof. Dr. Dr., Institut für Anatomie, Lehrstuhl für Anatomie und Entwicklungsbiologie, Univer-sitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany. Telephone: 49-201-7234380; Fax: 49-201-7235916; e-mail denker{at}uni-essen.de
Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc-like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial–mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E-cadherin and ZO-1 in the lower layer. In support of this, we found a concomitant 10-fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.
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