Differentiation of endothelial cells derived from mouse embryoid bodies: a possible in vitro vasculogenesis model.
Mouse embryonic stem cells (mES cells), which are pluripotent and self-renewal cells, are derived from the inner cell mass of mouse blastocysts. The objective of this study was to construct more efficient mES cell-derived embryoid bodies (EBs) for use as a vasculogenesis model and as an in vitro vascular toxicity testing model. EBs were formed for 3 days using hanging drop cultures and plated on gelatin-coated plates in endothelial growth medium-2 (EGM-2) to promote vascular development. The differentiation of mES cell-derived EBs was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and flow cytometry within 7 days after plating EBs. The mRNA and protein expressions of vascular endothelial growth factor receptors-2 (FLK-1), platelet endothelial cell adhesion molecule (PECAM), and vascular endothelial-cadherin (VE-cadherin) were observed in differentiated mES cells. When placed in matrigel, mES cell-derived endothelial like cells formed networks similar to vascular structures. mES cells were also exposed to 5-fluorouracil (5-FU), a strong inhibitor of vessel formation, and its cytotoxicity was determined using MTT assays. The inhibitory concentrations (IC50) of 5-FU for mES cells and C166 cells were 0.72 microM and 1.04 microM, respectively. These results demonstrate that mES cells can be used to study vasculogenesis and for cytotoxicity screening.
Kim
Gi Dae
GD
Department of Reproductive and Developmental Toxicology, National Institute of Toxicological Research, KFDA, Seoul 122-704, Republic of Korea.
Kim
Gi Jin
GJ
Seok
Ji Hyun
JH
Chung
Hyung-Min
HM
Chee
Kew-Mahn
KM
Rhee
Gyu-Seek
GS
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D002196:Capillaries; D002454:Cell Differentiation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000187:drug effects*; D042783:Endothelial Cells / Q000187:drug effects*; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology; D007150:Immunohistochemistry; D007797:Laminin; D051379:Mice; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D011509:Proteoglycans; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000964:Antimetabolites, Antineoplastic; D003470:Culture Media; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D012313:RNA; D003094:Collagen; D005472:Fluorouracil