Directed Cardiomyocyte Differentiation From Human Pluripotent Stem Cells by Modulating Wnt/β-catenin Signaling Under Fully Defined Conditions

Nat Protoc. 2013 Jan;8(1):162-75. doi: 10.1038/nprot.2012.150. Epub 2012 Dec 20.

Abstract

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor- and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of β-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8-1.3 million cardiomyocytes per cm(2)) of virtually pure (80-98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening and cell-based therapeutic applications.

Publication types

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

MeSH terms

  • Cell Culture Techniques*
  • Cell Differentiation*
  • Flow Cytometry
  • Humans
  • Lentivirus / metabolism
  • Myocytes, Cardiac / cytology*
  • Pluripotent Stem Cells / cytology*
  • RNA, Small Interfering / metabolism
  • Wnt Signaling Pathway*
  • beta Catenin / metabolism

Substances

  • RNA, Small Interfering
  • beta Catenin