Differentiation of human embryonic stem cells to cardiomyocytes on microcarrier cultures

Curr Protoc Stem Cell Biol. 2012 May:Chapter 1:Unit1D.7. doi: 10.1002/9780470151808.sc01d07s21.

Abstract

We have developed an improved cardiomyocyte differentiation protocol where we stabilized embryoid bodies (EB) in serum- and insulin-free medium (bSFS) supplemented with p38 MAP kinase inhibitor (SB203580) by addition of 10 µm laminin-coated positively charged (protamine sulfate derivatized TSKgel Tresyl-5PW) microcarriers. This protocol achieved a maximum 3-fold cell expansion, differentiation efficiency of 20%, and an overall cardiomyocyte yield of 3 × 10⁵ CM/ml in static conditions. In comparison, EB cultures achieved 1.5-fold cell expansion, differentiation efficiency of 15%, and an overall cardiomyocyte yield of 1.1 × 10⁵ CM/ml. The scalability of this platform was demonstrated in suspended spinner cultures, producing a maximum of 2.14 × 10⁵ CM/ml in 50-ml cultures. This yield is two-fold higher than the control static EB-based platform (1.1 × 10⁵ CM/ml), and seven-fold higher than yields reported in literature, 3.1-9 × 10⁴ CM/ml. The robustness of this protocol was tested with HES-3 and H1 cell lines.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Adhesion / drug effects
  • Cell Culture Techniques / methods*
  • Cell Differentiation* / drug effects
  • Cells, Cultured
  • Embryonic Stem Cells / cytology*
  • Embryonic Stem Cells / drug effects
  • Flow Cytometry
  • Humans
  • Laminin / pharmacology
  • Mice
  • Microspheres*
  • Myocytes, Cardiac / cytology*
  • Myocytes, Cardiac / drug effects

Substances

  • Laminin