EMBRYONIC STEM-CELLS DIFFERENTIATE IN-VITRO TO ENDOTHELIAL-CELLS THROUGH SUCCESSIVE MATURATION STEPS

The mechanisms involved in the regulation of vasculogenesis still rema in unclear in mammals. Totipotent embryonic stem (ES) cells may repres ent a suitable in vitro model to study molecular events involved in va scular development, In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-deriv ed embryoid bodies (EBs). Results of both reverse transcription-polyme rase chain reaction and/or immunofluorescence analysis show that a spo ntaneous endothelial differentiation occurs during EBs development. ES -derived endothelial cells express a full range of cell lineage-specif ic markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1 , tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-1 4.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation, PECA M and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5, Immunoflu orescence stainings of EBs with antibodies directed against Flk-1, PEC AM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression o f these antigens occurs at different steps of endothelial cell differe ntiation. The addition of an angiogenic growth factor mixture includin g erythropoietin, interleukin-6, fibroblast growth factor 2, and vascu lar endothelial growth factor in the EB culture medium significantly i ncreased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculo genesis. (C) 1996 by The American Society of Hematology.