Highly efficient and feeder-free production of subculturable vascular endothelial cells from primate embryonic stem cells.
The vascular endothelial cell (VEC) differentiation from primate embryonic stem (ES) cells has critical problems: low differentiation efficiencies (<2%) and/or subculture incapability. We report a novel feeder-free culture method for high efficiency production of subculturable VECs from cynomolgus monkey ES cells. Spheres, which were generated from ES cells in the presence of cytokine cocktail, were cultured on gelatin-coated plates. Cobblestone-shaped cells spread out after a few days, which were followed by an emergence of a sac-like structure containing hematopoietic cells. All adherent cells including sac walls cells and surrounding cobblestone cells expressed vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Subculture of these cells resulted in a generation of homogeneous spindle-shaped population bearing cord-forming activities and a uniform acetylated low density lipoprotein-uptaking capacity with von Willbrand factor and endothelial nitric oxide synthetase expressions. They were freeze-thaw-tolerable and subculturable up to eight passages. Co-existence of pericytes or immature ES cells was ruled out. When introduced in a collagen sponge plug implanted intraperitoneally in mice, ES-derived cells recruited into neovascularity. Although percentages of surface VE-cadherin-positive population varied from 20% to 80% as assessed by flow cytometry, the surface VE-cadherin-negative population showed intracellular VE-cadherin expression and mature functions, as we call it as atypical VECs. When sorted, the surface VE-cadherin-positive population expanded as almost pure (>90%) VE-cadherin/PECAM-1-positive VECs by 160-fold after five passages. Thus, our system provides pure production of functional, subculturable and freeze-thaw-tolerable VECs, including atypical VECs, from primate ES cells.
Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan.
Kumiko
K
Saeki
Yoshiko
Y
Yogiashi
Masako
M
Nakahara
Naoko
N
Nakamura
Satoko
S
Matsuyama
Akemi
A
Koyanagi
Hideo
H
Yagita
Makoto
M
Koyanagi
Yasushi
Y
Kondo
Akira
A
Yuo
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D007150:Immunohistochemistry; D008252:Macaca fascicularis; D020133:Reverse Transcriptase Polymerase Chain Reaction