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Culture and Preparation of Human Embryonic Stem Cells for Proteomics-Based Applications

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Human Embryonic Stem Cell Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 584))

Abstract

New challenges will arise as research into human embryonic stem (hES) cell differentiation moves from optimization and overcoming technical hurdles to mechanistic considerations. An immediate need will be to culture hES cells in the absence of contaminating feeder layers and allow for the preparation of purified DNA, RNA, and proteins to analyze changes in microRNA levels, gene expression, protein expression, and signal transduction. Purified, uniform populations of hES cells will allow researchers to better explore the biochemical mechanisms by which differentiation occurs.

Much recent work has focused upon genetic analysis of different stem cell populations. Expected variabilities between pluripotent hES cells, mesoderm, ectoderm, and definitive endoderm have been observed in microarray profiles (1–7). Interestingly, there also appears to be significant heterogeneity in mRNA expressed in different hES cell lines (8, 9). One approach to better understand how changes in mRNA levels in differentiating stem cells and individual hES cell lines relate to cell function is to study changes in signal transduction and global changes in protein expression. This chapter describes the methods routinely employed to prepare cells for analysis by traditional biochemistry (fractionation and western blotting) and proteomic analysis (2D electrophoresis/mass spectrometry and free-flow isoelectric focusing).

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Acknowledgements

The authors would like to thank Benjamin Lu for help developing methods for free-flow isoelectric focusing. This work was supported by the Larry L. Hillblom Foundation.

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Authors and Affiliations

  1. Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA

    Charles C. King

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  1. Charles C. King
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Editor information

Editors and Affiliations

  1. Sprott Centre for Stem Cell Research, Ottawa Hosp. Research Institute (OHRI), Smyth Road 501, Ottawa, K1H 8L6, Canada

    Kursad Turksen

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC 2006

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King, C.C. (2009). Culture and Preparation of Human Embryonic Stem Cells for Proteomics-Based Applications. In: Turksen, K. (eds) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 584. Humana Press. https://doi.org/10.1007/978-1-60761-369-5_9

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  • DOI: https://doi.org/10.1007/978-1-60761-369-5_9

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-368-8

  • Online ISBN: 978-1-60761-369-5

  • eBook Packages: Springer Protocols

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