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Highly efficient and feeder‐free production of subculturable vascular endothelial cells from primate embryonic stem cells
Author(s) -
Saeki Kumiko,
Yogiashi Yoshiko,
Nakahara Masako,
Nakamura Naoko,
Matsuyama Satoko,
Koyanagi Akemi,
Yagita Hideo,
Koyanagi Makoto,
Kondo Yasushi,
Yuo Akira
Publication year - 2008
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.21502
Subject(s) - population , microbiology and biotechnology , biology , stem cell , subculture (biology) , embryonic stem cell , ve cadherin , haematopoiesis , immunology , cell , cadherin , biochemistry , medicine , botany , environmental health , gene
Abstract 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. J. Cell. Physiol. 217: 261–280, 2008. © 2008 Wiley‐Liss, Inc.