Maintenance of Human Embryonic Stem Cells by Sphingosine-1-Phosphate and Platelet-Derived Growth Factor
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 wordsHuman embryonic stem cells Platelet-derived growth factor Serum-free medium Sphingosine-1-phosphate
R.C.B. Wong is supported by the California Institute of Regenerative Medicine Grant RC1-00110-1. A.P. is supported by a NHMRC/Victorian Neurotrauma Initiative Career Development Award.
- 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 37:1099–1103PubMedCrossRefGoogle Scholar
- 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 10:461–466PubMedCrossRefGoogle Scholar