title: Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions.
abstract: BACKGROUND
  Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage, differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally, current differentiation strategies are hampered by inefficiency, lack of reproducibility, and use of animal-derived products.
  
  
  METHODS
  To direct the differentiation of hESCs to endothelial subtypes, H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3), basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation, the endothelial progenitor cells (CD34(+)CD31(+) cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized.
  
  
  RESULTS
  Sequential modulation of hESCs with GSK-3 inhibitor, bFGF, BMP4 and VEGF resulted in stages reminiscent of primitive streak, early mesoderm/lateral plate mesoderm, and endothelial progenitors under feeder- and serum-free conditions. Furthermore, these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically, the endothelial progenitors differentiated to venous ECs in the absence of VEGF, and to arterial phenotype under low concentrations of VEGF. Additionally, these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse.
  
  
  CONCLUSIONS
  We report a simple, rapid, and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial-venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future.
authors.affiliation: Oral Sciences Disciplines, Faculty of Dentistry, National University of Singapore, Singapore, 119083, Singapore. sriramgopu@u.nus.edu.|Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Groove, #06-06 Immunos, Singapore, 138648, Singapore. sriramgopu@u.nus.edu.
authors.forename: Gopu
authors.initials: G
authors.lastname: Sriram
authors.affiliation: Oral Sciences Disciplines, Faculty of Dentistry, National University of Singapore, Singapore, 119083, Singapore. jiayong.tan@cardinalhealth.com.
authors.forename: Jia Yong
authors.initials: JY
authors.lastname: Tan
authors.affiliation: Oral and Maxillofacial Surgery Disciplines, Faculty of Dentistry, National University of Singapore, Singapore, 119083, Singapore. denii@nus.edu.sg.
authors.forename: Intekhab
authors.initials: I
authors.lastname: Islam
authors.affiliation: Cardiac, Thoracic and Vascular Surgery (CTVS) Laboratory, Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117510, Singapore. surraj@nus.edu.sg.|Singapore-Technion Alliance For Research and Technology (START) Regenerative Medicine Laboratory, Campus for Research Excellence And Technological Enterprise (CREATE), Singapore, 138602, Singapore. surraj@nus.edu.sg.
authors.forename: Abdul Jalil
authors.initials: AJ
authors.lastname: Rufaihah
authors.affiliation: Oral Sciences Disciplines, Faculty of Dentistry, National University of Singapore, Singapore, 119083, Singapore. dencaot@nus.edu.sg.|NUS Graduate School for Integrative Science and Engineering, Singapore, 117456, Singapore. dencaot@nus.edu.sg.|Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore, 117456, Singapore. dencaot@nus.edu.sg.
authors.forename: Tong
authors.initials: T
authors.lastname: Cao
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D001158:Arteries / Q000166:cytology; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D004730:Endothelium, Vascular / Q000166:cytology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D000066449:Human Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D014183:Transplantation, Heterologous; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; D055415:Bone Morphogenetic Protein 4; C473711:Chir 99021; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011725:Pyridines; D011743:Pyrimidines; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D038362:Glycogen Synthase Kinase 3