Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures

Stem Cells. Jun-Jul 2005;23(6):772-80. doi: 10.1634/stemcells.2004-0184.

Abstract

Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is low. We routinely induce cardiomyocyte differentiation of the HES-2 cell line by coculture with a visceral endoderm-like cell line, END-2, in the presence of 20% fetal calf serum (FCS). In this study, we demonstrate a striking inverse relationship between cardiomyocyte differentiation and the concentration of FCS during HES-2-END-2 coculture. The number of beating areas in the cocultures was increased 24-fold in the absence of FCS compared with the presence of 20% FCS. An additional 40% increase in the number of beating areas was observed when ascorbic acid was added to serum-free cocultures. The increase in serum-free cocultures was accompanied by increased mRNA and protein expression of cardiac markers and of Isl1, a marker of cardiac progenitor cells. The number of beating areas increased up to 12 days after initiation of coculture of HES-2 with END-2 cells. However, the number of alpha-actinin-positive cardiomyocytes per beating area did not differ significantly between serum-free cocultures (503 +/- 179; mean +/- standard error of the mean) and 20% FCS cocultures (312 +/- 227). The stimulating effect of serum-free coculture on cardiomyocyte differentiation was observed not only in HES-2 but also in the HES-3 and HES-4 cell lines. To produce sufficient cardiomyocytes for cell replacement therapy in the future, upscaling cardiomyocyte formation from hESCs is essential. The present data provide a step in this direction and represent an improved in vitro model, without interfering factors in serum, for testing other factors that might promote cardiomyocyte differentiation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinin / metabolism
  • Animals
  • Blotting, Western
  • Cell Culture Techniques / methods
  • Cell Differentiation
  • Cell Line
  • Cells, Cultured
  • Coculture Techniques
  • Culture Media, Serum-Free / pharmacology
  • Embryo, Mammalian / cytology*
  • Humans
  • Immunohistochemistry
  • Mice
  • Microscopy, Fluorescence
  • Myocytes, Cardiac / cytology*
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Stem Cells / cytology*

Substances

  • Culture Media, Serum-Free
  • RNA, Messenger
  • Actinin