[Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].
OBJECTIVE
To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo.


METHODS
Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed.


RESULTS
The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4.


CONCLUSIONS
The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.
Jiang
Hua
H
Obstetrics & Gynecology Hospital, Fudan University, Shanghai 200011, China.
Feng
You-Ji
YJ
Xie
Yi
Y
Han
Jin-Lan
JL
Wang
Zack
Z
Chen
Tong
T
D001808:Blood Vessels / Q000196:embryology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D047108:Embryonic Development; D053595:Embryonic Stem Cells / Q000166:cytology*; D042783:Endothelial Cells / Q000166:cytology; D006801:Humans; D008954:Models, Biological; D038081:Organogenesis