title: Derivation of endothelial cells from human embryonic stem cells in fully defined medium enables identification of lysophosphatidic acid and platelet activating factor as regulators of eNOS localization.
abstract: The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34(+)KDR(+) endothelial progenitor cells after 6days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization.
authors.affiliation: Monash Immunology and Stem Cell Laboratories (MISCL), Monash University, Clayton, Victoria, Australia. magdaline.costa@monash.edu
authors.forename: Magdaline
authors.initials: M
authors.lastname: Costa
authors.forename: Koula
authors.initials: K
authors.lastname: Sourris
authors.forename: Sue Mei
authors.initials: SM
authors.lastname: Lim
authors.forename: Qing C
authors.initials: QC
authors.lastname: Yu
authors.forename: Claire E
authors.initials: CE
authors.lastname: Hirst
authors.forename: Helena C
authors.initials: HC
authors.lastname: Parkington
authors.forename: Vanta J
authors.initials: VJ
authors.lastname: Jokubaitis
authors.forename: Anthony E
authors.initials: AE
authors.lastname: Dear
authors.forename: Hong B
authors.initials: HB
authors.lastname: Liu
authors.forename: Suzanne J
authors.initials: SJ
authors.lastname: Micallef
authors.forename: Kathy
authors.initials: K
authors.lastname: Koutsis
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Elefanty
authors.forename: Edouard G
authors.initials: EG
authors.lastname: Stanley
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D003094:Collagen / Q000737:chemistry; D003470:Culture Media / Q000494:pharmacology*; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects*; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D020869:Gene Expression Profiling; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007797:Laminin / Q000737:chemistry; D008246:Lysophospholipids / Q000494:pharmacology*; D052250:Nitric Oxide Synthase Type III / Q000032:analysis* / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology*; D011509:Proteoglycans / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; D003470:Culture Media; D004338:Drug Combinations; D007797:Laminin; D008246:Lysophospholipids; D010972:Platelet Activating Factor; D011509:Proteoglycans; D014409:Tumor Necrosis Factor-alpha; C059485:matrigel; D003094:Collagen; D052250:Nitric Oxide Synthase Type III; C032881:lysophosphatidic acid