Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: the matrix sandwich method

Circ Res. 2012 Oct 12;111(9):1125-36. doi: 10.1161/CIRCRESAHA.112.273144. Epub 2012 Aug 21.


Rationale: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation.

Objective: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis.

Methods and results: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms.

Conclusions: Dynamic extracellular matrix application promoted epithelial-mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Publication types

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

MeSH terms

  • Activins / pharmacology
  • Bone Morphogenetic Protein 4 / pharmacology
  • Cell Culture Techniques / methods*
  • Cell Differentiation / drug effects
  • Cell Differentiation / physiology*
  • Cell Line
  • Cells, Cultured
  • Collagen*
  • Drug Combinations
  • Epithelial-Mesenchymal Transition / drug effects
  • Epithelial-Mesenchymal Transition / physiology
  • Extracellular Matrix / physiology*
  • Fibroblast Growth Factor 2 / pharmacology
  • Humans
  • Intercellular Signaling Peptides and Proteins / pharmacology
  • Laminin*
  • Myocytes, Cardiac / cytology*
  • Myocytes, Cardiac / drug effects
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / drug effects
  • Proteoglycans*
  • Signal Transduction / drug effects
  • Signal Transduction / physiology


  • BMP4 protein, human
  • Bone Morphogenetic Protein 4
  • Drug Combinations
  • Intercellular Signaling Peptides and Proteins
  • Laminin
  • Proteoglycans
  • activin A
  • Fibroblast Growth Factor 2
  • Activins
  • matrigel
  • Collagen