An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. I: Establishment of the differentiation protocol.
The aim of the present study was to establish an experimental protocol to differentiate murine embryonic stem (ES) cells into endothelial cells in vitro. The spinner flask technique as well as the hanging drop method were used to generate so-called embryoid bodies (EBs). In order to find out the optimal differentiation environment, EBs were cultured under various experimental conditions for up to 14 days. The influence of basic fibroblast growth factor (bFGF) alone, vascular endothelial growth factor (VEGF) alone, bFGF and VEGF together and a cocktail consisting of bFGF, VEGF, interleukin-6 (IL-6) and erythropoietin (Epo) on the induction of differentiation of ES cells into endothelial cells was studied. Different concentrations of growth factors and times of treatment were applied. Endothelial cells were characterized by analyzing the expression of platelet-endothelial cell adhesion molecule (PECAM-1), the endothelial-specific vascular endothelial cadherin (VE-Cadherin), the angiopoietin receptor Tie-2, VEGF receptors 1 and 2 (Flt-1 and Flk-1, respectively) and the soluble form of Flt-1 (sFlt) at the mRNA level. PECAM-1 and VE-Cadherin were also studied at the protein level. The data clearly showed that EBs generated by the hanging drop method, followed by their transfer into suspension culture on day 3 of differentiation and their subsequent plating on day 5 is the best of the studied methods to differentiate ES cells into endothelial cells. Addition of VEGF alone or a cocktail consisting of VEGF, bFGF, IL-6 and Epo resulted in the strongest gene expression levels of the above mentioned endothelial cell markers in the differentiated ES cells.
Festag
Matthias
M
Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, 88397 Biberach an der Riss, Germany. matthias.festag@roche.com
Sehner
Claudia
C
Steinberg
Pablo
P
Viertel
Bruno
B
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects*; D042783:Endothelial Cells / Q000166:cytology* / Q000502:physiology; D005786:Gene Expression Regulation; D051379:Mice; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D042787:Receptor, TIE-2 / Q000235:genetics / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D003470:Culture Media; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D042787:Receptor, TIE-2; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099152:Cadherin 5; C501163:Flt1 protein, mouse