[In vitro vasculogenesis and angiogenesis of mouse embryonic stem cells].
OBJECTIVE
To explore an optional condition to induce mouse embryonic stem (ES) cells to differentiate into endothelial cells and to establish in vitro models of vasculogenesis and angiogenesis.


METHODS
Mouse ES cells were cultured in differentiation medium containing a cocktail of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-6 (IL-6) and erythropoietin (EPO) in 1% methylcellulose to induce formation of embryoid bodies (EBs). At day 11, EBs were harvested and suspended in rat-tail collagen type I with the same cocktail of cytokines cultured for three additional days. The differentiation of ES cells into endothelial cells, processes of vasculogenesis and angiogenesis were examined using immunostaining of EBs slices and whole-mount immunocytochemistry of EBs with monoclonal antibodies (mAbs) against platelet endothelial cell adhesion molecule-1 (PECAM-1) and alpha-smooth muscle actin (SMA).


RESULTS
Under appropriate culture conditions; ES cells spontaneously differentiated and formed EBs containing vascular structures and tubular channels, which were positive for PECAM-1 co-differentiated with smooth muscle. When not treated with angiogenic growth factors, PECAM-1-positive cells could not organize into vascular structures of 11-day-old EBs. In the presence of angiogenic factors 11-day old EBs embedded into type I collagen, and rapidly developed an endothelial networks. Whole-mount immunocytochemistry of collagen gel with anti-PECAM-1 antibody showed the formation of primary vascular structures sprouting from EBs. Quantitative analysis revealed that 100 microg/ml thalidomide significantly reduced the number and length of EBs endothelial sprouting.


CONCLUSIONS
Mouse ES cells can differentiate into endothelial cells combined with smooth muscle differentiation during EBs formation and further develop endothelial outgrowths after EBs embedded into collagen, which respectively recapitulate vasculogenesis, angiogenesis, and arteriogenesis processes in vivo. The models provide a useful tool to investigate vasculogenesis, angiogenesis, and arteriogenesis mechanisms and evaluate the effects of angiogenic and angiostatic agents.
Li
Zong-jin
ZJ
State Key Laboratory of Experimental Hematology, Institute of Hematology, CAMS and PUMC, Tianjin 300020, China.
Yang
Chen
C
Tang
Feng-wu
FW
Zhang
Zhi-hua
ZH
Xu
Bin
B
Zhao
Qin-jun
QJ
Yang
Ren-chi
RC
Wang
Zheng-yu
ZY
Han
Zhong-chao
ZC
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D003094:Collagen / Q000494:pharmacology; D003470:Culture Media; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D004921:Erythropoietin / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D051379:Mice; D018919:Neovascularization, Physiologic / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D003470:Culture Media; D015850:Interleukin-6; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D004921:Erythropoietin; D003094:Collagen