title: VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering.
abstract: OBJECTIVE
  Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs.
  
  
  RESULTS
  To enhance endothelial cell differentiation above a baseline of approximately 2% in embryoid body (EB) spontaneous differentiation, 3 alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10 to 14. Continuous VEGF treatment resulted in a 4- to 5-fold enrichment of CD31(+) cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31(+) cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFalpha, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells.
  
  
  CONCLUSIONS
  VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. This enrichment method increases endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants.
authors.affiliation: Arra and Eva Woods Professor of Pathology and Bioengineering, Director, Center for Cardiovascular Biology, Codirector, Institute for Stem Cell and Regenerative Medicine, University of Washington, 815 Mercer Street, Room 453 Brotman Building, Seattle, WA 98109, USA.
authors.forename: Marilyn B
authors.initials: MB
authors.lastname: Nourse
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Halpin
authors.forename: Marta
authors.initials: M
authors.lastname: Scatena
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Mortisen
authors.forename: Nathaniel L
authors.initials: NL
authors.lastname: Tulloch
authors.forename: Kip D
authors.initials: KD
authors.lastname: Hauch
authors.forename: Beverly
authors.initials: B
authors.lastname: Torok-Storb
authors.forename: Buddy D
authors.initials: BD
authors.lastname: Ratner
authors.forename: Lil
authors.initials: L
authors.lastname: Pabon
authors.forename: Charles E
authors.initials: CE
authors.lastname: Murry
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D003094:Collagen; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007797:Laminin; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology; D011509:Proteoglycans; D051381:Rats; D011923:Rats, Nude; D023822:Tissue Engineering / Q000379:methods*; D020298:U937 Cells; D014471:Umbilical Veins / Q000166:cytology; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D015415:Biomarkers; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C467484:VEGFA protein, human; D019010:Vascular Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; C059485:matrigel; D018799:Intercellular Adhesion Molecule-1; D003094:Collagen