Protocol

Part of the series Methods in Molecular Biology pp 1-8

Date:

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

  • Raymond C. B. WongAffiliated withCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, The University of MelbourneOphthalmology, Department of Surgery, The University of Melbourne
  • , Martin F. PeraAffiliated withDepartment of Anatomy and Neurosciences, Florey Neuroscience and Mental Health Institute, Walter and Eliza Hall Institute of Medical Research, The University of Melbourne
  • , Alice PébayAffiliated withCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, The University of MelbourneOphthalmology, Department of Surgery, The University of Melbourne Email author 

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Abstract

Human embryonic stem cells (hESCs) have historically been cultivated on feeder layers of primary mouse embryonic fibroblasts (MEF) in a medium 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 hESCs in culture by addition of sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF). This complete protocol provides a simple alternative chemically defined serum-free system that is relatively inexpensive and advantageous for studying signaling pathways involved in hESC pluripotency.

Keywords:

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