title: A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.
abstract: BACKGROUND
  The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34(+) cord blood using non-viral, non-integrating methods.
  
  
  RESULTS
  We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+) cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89%) of cardiac troponin I(+) cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.
  
  
  CONCLUSIONS
  This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.
authors.affiliation: Johns Hopkins Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America. paul.burridge@jhmi.edu
authors.forename: Paul W
authors.initials: PW
authors.lastname: Burridge
authors.forename: Susan
authors.initials: S
authors.lastname: Thompson
authors.forename: Michal A
authors.initials: MA
authors.lastname: Millrod
authors.forename: Seth
authors.initials: S
authors.lastname: Weinberg
authors.forename: Xuan
authors.initials: X
authors.lastname: Yuan
authors.forename: Ann
authors.initials: A
authors.lastname: Peters
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Mahairaki
authors.forename: Vassilis E
authors.initials: VE
authors.lastname: Koliatsos
authors.forename: Leslie
authors.initials: L
authors.lastname: Tung
authors.forename: Elias T
authors.initials: ET
authors.lastname: Zambidis
mesh_terms: D000328:Adult; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D019521:Body Patterning / Q000187:drug effects; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D055724:Electrophysiological Phenomena / Q000187:drug effects; D058732:Embryoid Bodies / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology; D011142:Polyvinyl Alcohol / Q000494:pharmacology; D019076:Transgenes / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D055415:Bone Morphogenetic Protein 4; D003470:Culture Media; D007328:Insulin; D016222:Fibroblast Growth Factor 2; D011142:Polyvinyl Alcohol; D010100:Oxygen