title: Evaluation of the use of an induced puripotent stem cell sheet for the construction of tissue-engineered vascular grafts.
abstract: OBJECTIVE
  The development of a living, tissue-engineered vascular graft (TEVG) holds great promise for advancing the field of cardiovascular surgery. However, the ultimate source and time needed to procure these cells remain problematic. Induced puripotent stem (iPS) cells have recently been developed and have the potential for creating a pluripotent cell line from a patient's own somatic cells. In the present study, we evaluated the use of a sheet created from iPS cell-derived vascular cells as a potential source for the construction of TEVG.
  
  
  METHODS
  Male mouse iPS cells were differentiated into embryoid bodies using the hanging-drop method. Cell differentiation was confirmed by a decrease in the proportion of SSEA-1-positive cells over time using fluorescence-activated cell sorting. The expression of endothelial cell and smooth muscle cell markers was detected using real-time polymerase chain reaction (PCR). The differentiated iPS cell sheet was made using temperature-responsive dishes and then seeded onto a biodegradable scaffold composed of polyglycolic acid-poly-l-lactide and poly(l-lactide-co-ε-caprolactone) with a diameter of 0.8 mm. These scaffolds were implanted as interposition grafts in the inferior vena cava of female severe combined immunodeficiency/beige mice (n = 15). Graft function was serially monitored using ultrasonography. The grafts were analyzed at 1, 4, and 10 weeks with histologic examination and immunohistochemistry. The behavior of seeded differentiated iPS cells was tracked using Y-chromosome fluorescent in situ hybridization and SRY real-time PCR.
  
  
  RESULTS
  All mice survived without thrombosis, aneurysm formation, graft rupture, or calcification. PCR evaluation of iPS cell sheets in vitro demonstrated increased expression of endothelial cell markers. Histologic evaluation of the grafts demonstrated endothelialization with von Willebrand factor and an inner layer with smooth muscle actin- and calponin-positive cells at 10 weeks. The number of seeded differentiated iPS cells was found to decrease over time using real-time PCR (42.2% at 1 week, 10.4% at 4 weeks, 9.8% at 10 weeks). A fraction of the iPS cells were found to be Y-chromosome fluorescent positive at 1 week. No iPS cells were found to co-localize with von Willebrand factor or smooth muscle actin-positive cells at 10 weeks.
  
  
  CONCLUSIONS
  Differentiated iPS cells offer an alternative cell source for constructing TEVG. Seeded iPS cells exerted a paracrine effect to induce neotissue formation in the acute phase and were reduced in number by apoptosis at later time points. Sheet seeding of our TEVG represents a viable mode of iPS cell delivery over time.
authors.affiliation: Section of Cardiac Surgery, Yale University School of Medicine, New Haven, CT 06510, USA.
authors.forename: Narutoshi
authors.initials: N
authors.lastname: Hibino
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Duncan
authors.forename: Ani
authors.initials: A
authors.lastname: Nalbandian
authors.forename: Tai
authors.initials: T
authors.lastname: Yi
authors.forename: Yibing
authors.initials: Y
authors.lastname: Qyang
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Shinoka
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Breuer
mesh_terms: D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D001807:Blood Vessel Prosthesis; D019917:Blood Vessel Prosthesis Implantation / Q000295:instrumentation*; D002454:Cell Differentiation; D002460:Cell Line; D005260:Female; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism / Q000637:transplantation*; D019344:Lactic Acid / Q000737:chemistry; D008297:Male; D051379:Mice; D016513:Mice, SCID; D019899:Paracrine Communication; D011091:Polyesters / Q000737:chemistry; D011100:Polyglycolic Acid / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011474:Prosthesis Design; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds; D014463:Ultrasonography; D014682:Vena Cava, Inferior / Q000000981:diagnostic imaging / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D014998:Y Chromosome / Q000378:metabolism
chemical_list: D015415:Biomarkers; D011091:Polyesters; D011108:Polymers; D011100:Polyglycolic Acid; D019344:Lactic Acid; C033616:poly(lactide); C062968:lactide-caprolactone copolymer