title: Serum-free differentiation of functional human coronary-like vascular smooth muscle cells from embryonic stem cells.
abstract: OBJECTIVE
  Despite the diverse developmental origins of vascular smooth muscle cells (VSMCs), recent attempts to generate VSMCs from human embryonic stem cells (hESCs) differentiated along various lineages did not yield distinct cell phenotypes. The aim of this study was to derive and characterize functional coronary-like VSMCs from hESCs using serum-free cardiac-directed differentiation.
  
  
  RESULTS
  Embryoid bodies (EBs) from three pluripotent stem cell lines subjected to cardiac-directed differentiation in defined media were characterized over 30 days for VSMC-specific gene expression by qRT-PCR, immunofluorescence microscopy and fluorescence-activated cell sorting (FACS). EBs composed of cardiomyocytes, endothelial cells (ECs), fibroblasts, and VSMCs underwent FACS on d28 to reveal that the VSMCs form a distinct subpopulation, which migrate with ECs in an in vitro angiogenesis assay. To enrich for VSMCs, d28 EBs were dissociated and cultured as monolayers. Over several passages, mRNA and protein levels of cardiomyocyte, endothelial, and fibroblast markers were abolished, whereas those of mature VSMCs were unchanged. Vascular endothelial growth factor and basic fibroblast growth factor were critical for the separation of the cardiac and VSMC lineages in EBs, and for the enrichment of functional VSMCs in monolayer cultures. Calcium cycling and cell shortening responses to vasoconstrictors in hESC-derived VSMCs in vitro were indistinguishable from primary human coronary artery SMCs, and distinct from bladder and aorta SMCs. VSMCs identically derived from green fluorescent protein -expressing hESCs integrated in and contributed to new vessel formation in vivo.
  
  
  CONCLUSIONS
  The ability to generate hESC-derived functional human coronary-like VSMCs in serum-free conditions has implications for disease modelling, drug screening, and regenerative therapies.
authors.affiliation: McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada.
authors.forename: Omar
authors.initials: O
authors.lastname: El-Mounayri
authors.forename: Anton
authors.initials: A
authors.lastname: Mihic
authors.forename: Eric A
authors.initials: EA
authors.lastname: Shikatani
authors.forename: Mark
authors.initials: M
authors.lastname: Gagliardi
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Steinbach
authors.forename: Nicole
authors.initials: N
authors.lastname: Dubois
authors.forename: Ralph
authors.initials: R
authors.lastname: Dacosta
authors.forename: Ren-Ke
authors.initials: RK
authors.lastname: Li
authors.forename: Gordon
authors.initials: G
authors.lastname: Keller
authors.forename: Mansoor
authors.initials: M
authors.lastname: Husain
mesh_terms: D000199:Actins / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology*; D016895:Culture Media, Serum-Free; D053595:Embryonic Stem Cells / Q000166:cytology*; D016222:Fibroblast Growth Factor 2 / Q000502:physiology; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D018919:Neovascularization, Physiologic; D012333:RNA, Messenger / Q000032:analysis; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology; D014661:Vasoconstriction / Q000187:drug effects
chemical_list: D000199:Actins; D016895:Culture Media, Serum-Free; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D002118:Calcium