pp 1-8 | Cite as

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

  • Raymond C. B. Wong
  • Martin F. Pera
  • Alice Pébay
Protocol
Part of the Methods in Molecular Biology book series

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 

References

  1. 1.
    Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404CrossRefPubMedGoogle Scholar
  2. 2.
    Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147ADSCrossRefPubMedGoogle Scholar
  3. 3.
    Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 227:271–278CrossRefPubMedGoogle Scholar
  4. 4.
    Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA (2006) Feeder-independent culture of human embryonic stem cells. Nat Methods 3:637–646CrossRefPubMedGoogle Scholar
  5. 5.
    Pebay A, Wong RC, Pitson SM, Wolvetang EJ, Peh GS, Filipczyk A, Koh KL, Tellis I, Nguyen LT, Pera MF (2005) Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells. Stem Cells 23:1541–1548CrossRefPubMedGoogle Scholar
  6. 6.
    Wong RC, Tellis I, Jamshidi P, Pera M, Pebay A (2007) Anti-apoptotic effect of sphingosine-1-phosphate and platelet-derived growth factor in human embryonic stem cells. Stem Cells Dev 16:989–1001CrossRefPubMedGoogle Scholar
  7. 7.
    Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson J, Meisner L, Zwaka TP, Thomson JA, Andrews PW (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 22:53–54CrossRefPubMedGoogle Scholar
  8. 8.
    Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V, Kassauei K, Sui G, Cutler DJ, Liu Y, Brimble SN, Noaksson K, Hyllner J, Schulz TC, Zeng X, Freed WJ, Crook J, Abraham S, Colman A, Sartipy P, Matsui SI, Carpenter M, Gazdar AF, Rao M, Chakravarti A (2005) Genomic alterations in cultured human embryonic stem cells. Nat Genet 37:1099–1103CrossRefPubMedGoogle Scholar
  9. 9.
    Inzunza J, Sahlen S, Holmberg K, Stromberg AM, Teerijoki H, Blennow E, Hovatta O, Malmgren H (2004) Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol Hum Reprod 10:461–466CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Raymond C. B. Wong
    • 1
    • 3
  • Martin F. Pera
    • 2
  • Alice Pébay
    • 1
    • 3
  1. 1.Centre for Eye Research Australia, Royal Victorian Eye and Ear HospitalThe University of MelbourneEast MelbourneAustralia
  2. 2.Department of Anatomy and Neurosciences, Florey Neuroscience and Mental Health Institute, Walter and Eliza Hall Institute of Medical ResearchThe University of MelbourneMelbourneAustralia
  3. 3.Ophthalmology, Department of SurgeryThe University of MelbourneMelbourneAustralia

Personalised recommendations