High-efficiency production of subculturable vascular endothelial cells from feeder-free human embryonic stem cells without cell-sorting technique.
We previously reported a feeder-free culture method for pure production of subculturable vascular endothelial cells (VECs) from cynomolgus monkey embryonic stem cells (cmESCs) without as using cell-sorting technique. By this method, canonical vascular endothelial (VE)-cadherin/platelet-endothelial cell adhesion molecule 1 (PECAM1)-positive VECs (c-VECs) and atypical VE-cadherin/PECAM1-negative VECs (a-VECs) were generated without a contamination by pericytes, lymphatic endothelial cells, or immature ES cells. More recently, we established a unique culture technique to maintain human ESCs (hESCs) under a feeder-free and recombinant cytokine-free condition. Combining these two systems, we have successfully generated pure VECs from two lines of hESCs, khES-1 and khES-3, under a completely feeder-free condition. Our method is very simple: spheres generated from hESCs by floating culture using differentiation media supplemented with vascular endothelial growth factor, bone morphogenetic protein 4, stem cell factor, FMS-related tyrosine kinase-3 ligand, and interleukin 3 (IL3) and IL6 were cultured on gelatin-coated plates. Cell passage was performed by an ordinary enzymatic treatment. The hESC-derived differentiated cells demosntrated cord-forming activities and acetylated low-density lipoprotein-uptaking capacities. Moreover, they exclusively expressed von Willebrand factor and endothelial nitric oxide synthase. Flow cytometric analyses indicate that khES-3 generated both c-VECs and a-VECs as in the case of cmESCs. By contrast, khES-1 produced only a-VECs, which nonetheless demonstrated effective recruitment into neovascularity in vivo. Interestingly, a-VECs turned to express PECAM1 after transplantation into immunodeficient mice. The hESC-derived VECs were subculturable at least up to 10 passages without functional depression. Our method does not require a presorting processes to enrich progenitor fractions such as CD34-positive or kinase insert domain receptor (KDR)-positive cells, providing the most efficient and easiest technique for VEC production from hESCs.
Nakahara
Masako
M
Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan.
Nakamura
Naoko
N
Matsuyama
Satoko
S
Yogiashi
Yoshiko
Y
Yasuda
Kazuki
K
Kondo
Yasushi
Y
Yuo
Akira
A
Saeki
Kumiko
K
D015703:Antigens, CD / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D016895:Culture Media, Serum-Free; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D018919:Neovascularization, Physiologic / Q000502:physiology; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D000099152:Cadherin 5
D015703:Antigens, CD; D055415:Bone Morphogenetic Protein 4; D015820:Cadherins; D016895:Culture Media, Serum-Free; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099152:Cadherin 5