Abstract
By altering the cellular microenvironment and culture media composition, embryonic stem cells (ESCs) can be induced to differentiate in vitro into somatic cell types from the three primitive germ layers. ESC differentiation is regulated by an intricate series of signaling events that result in their transcriptional reprogramming, asymmetric cell division, and differentiation. Using various pharmacological agents and/or genetic manipulations, one can drive and enrich ESC differentiation to specific cell lineages. Identifying the transcriptional fingerprint during ESC differentiation could yield novel targets for genetic or pharmacological regulation to increase the yield of desirable cell types. We discuss here how to culture undifferentiated mouse ESCs (E14 line from 129/Ola) and generate embryoid bodies (EBs). We also discuss in detail how to prepare Affymetrix samples, how to hybridize and scan arrays, and how to quality control data and generate signal values and permutation based P-values.
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Acknowledgments
The authors would like to thank Drs. Whitmore Tingley and Roland Russnak for their contributions to the protocols for E14 ESC culture and embryoid body formations and Bruce Conklin and the late Karen Vranizan for their contributions to the design and interpretation of the data presented in this manuscript. We would also like to thank the Gladstone editorial department Gary Howard and Stephen Ordway for their contributions. Dedicated to the memory of Karen Vranizan.
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Zambon, A.C., Barker, C.S. (2010). Microarray Analysis of Embryonic Stem Cells and Differentiated Embryoid Bodies. In: Chittur, S.V. (eds) Microarray Methods for Drug Discovery. Methods in Molecular Biology, vol 632. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-663-4_3
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DOI: https://doi.org/10.1007/978-1-60761-663-4_3
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