Protocol

Sphingosine-1-Phosphate

Volume 874 of the series Methods in Molecular Biology pp 167-175

Date:

Maintenance of Human Embryonic Stem Cells by Sphingosine-1-Phosphate and Platelet-Derived Growth Factor

  • Raymond C. B. WongAffiliated withDepartment of Biological Chemistry, University of California Irvine
  • , Martin F. PeraAffiliated withEli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern CaliforniaCentre for Neuroscience, The University of Melbourne
  • , Alice PébayAffiliated withDepartment of Pharmacology, Centre for Neuroscience, The University of Melbourne Email author 

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Abstract

Embryonic stem cells are pluripotent and capable of indefinite self-renewal in vitro. Human embryonic stem cells (hESC) have generally been cultivated on feeder layers of primary mouse embryonic fibroblasts (MEF) in media supplemented with fetal calf serum (FCS). However, serum contains a wide variety of biologically active compounds that might adversely affect hESC growth and differentiation. Thus, cultivation of stem cells in FCS complicates experimental approaches to define the intracellular mechanisms required for hESC maintenance. This chapter describes the serum-free maintenance of hESC in culture by addition of sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF). This complete protocol provides a chemically defined serum-free system that is advantageous for studying signaling pathways involved in hESC pluripotency.

Key words

Human embryonic stem cells Platelet-derived growth factor Serum-free medium Sphingosine-1-phosphate