A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability

PLoS One. 2011 Apr 8;6(4):e18293. doi: 10.1371/journal.pone.0018293.

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.

Methodology/principal findings: 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.

Conclusion/significance: 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Antigens, CD34 / metabolism
  • Body Patterning / drug effects
  • Bone Morphogenetic Protein 4 / pharmacology
  • Cell Adhesion / drug effects
  • Cell Culture Techniques / methods*
  • Cell Differentiation* / drug effects
  • Cell Line
  • Cell Proliferation / drug effects
  • Culture Media / pharmacology
  • Electrophysiological Phenomena / drug effects
  • Embryoid Bodies / cytology
  • Embryoid Bodies / drug effects
  • Embryoid Bodies / metabolism
  • Fetal Blood / cytology
  • Fibroblast Growth Factor 2 / pharmacology
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Genetic Vectors / genetics
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / drug effects
  • Induced Pluripotent Stem Cells / metabolism
  • Insulin / pharmacology
  • Mesoderm / cytology
  • Mesoderm / drug effects
  • Mice
  • Myocytes, Cardiac / cytology*
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism
  • Oxygen / pharmacology
  • Polyvinyl Alcohol / pharmacology
  • Transgenes / genetics

Substances

  • Antigens, CD34
  • Bone Morphogenetic Protein 4
  • Culture Media
  • Insulin
  • Fibroblast Growth Factor 2
  • Polyvinyl Alcohol
  • Oxygen