ROCK suppression promotes differentiation and expansion of endothelial cells from embryonic stem cell-derived Flk1(+) mesodermal precursor cells.
Successful differentiation and expansion of endothelial cells (ECs) from embryonic stem cell (ESC)-derived Flk1(+) mesodermal precursor cells (MPCs) requires supplementation of vascular endothelial growth factor-A (VEGF-A). While analyzing VEGF-A/VEGFR2 downstream signaling pathway that underlies the VEGF-A-induced differentiation and expansion of ECs, we fortuitously found that Rho-associated protein kinase (ROCK) inhibitor Y27632 profoundly promoted the differentiation and expansion of ECs from Flk1(+) MPCs while reducing the differentiation and expansion of mural cells. The ROCK suppression-induced expansion of ECs appears to have resulted from promotion of proliferation of ECs via activation of PI3-kinase-Akt signaling. The ECs obtained by the combination of ROCK suppression and VEGF-A supplementation faithfully expressed most pan-EC surface makers, and phenotypic analyses revealed that they were differentiated toward arterial EC. Further incubation of the ICAM2(+) ECs with Y27632 and VEGF-A for 2 days promoted expansion of ECs by 6.5-fold compared with those incubated with only VEGF-A. Importantly, the ROCK suppression-induced ECs displayed neovasculogenic abilities in vitro and in vivo. Thus, supplementation of ROCK inhibitor Y27632 along with VEGF-A in 2D Matrigel culture system provides a simple, efficient, and versatile method for obtaining ample amount of ESC-derived ECs at high purity suitable for use in therapeutic neovascularization.
Laboratory for Vascular Biology and Stem Cell, Korea Advanced Institute of Science and Technology, Daejeon, Korea.
Hyung Joon
HJ
Joo
Dong-Kyu
DK
Choi
Joon Seo
JS
Lim
Jin-Sung
JS
Park
Seung-Hun
SH
Lee
Sukhyun
S
Song
Jennifer H
JH
Shin
Do-Sun
DS
Lim
Injune
I
Kim
Ki-Chul
KC
Hwang
Gou Young
GY
Koh
D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007797:Laminin / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018919:Neovascularization, Physiologic; D011509:Proteoglycans / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000577:Amides; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D007797:Laminin; D011509:Proteoglycans; D011725:Pyridines; D042461:Vascular Endothelial Growth Factor A; C059485:matrigel; C108830:Y 27632; D003094:Collagen; D040301:Vascular Endothelial Growth Factor Receptor-2; D054460:rho-Associated Kinases