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Gene Knockout Protocols pp 219–250Cite as

Differentiation Analysis of Pluripotent Mouse Embryonic Stem (ES) Cells In Vitro

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 530))

Abstract

Pluripotent embryonic stem (ES) cells are characterized by their almost unlimited potential to self-renew and to differentiate into virtually any cell type of the organism. Here we describe basic protocols for the in vitro differentiation of mouse ES cells into cells of the cardiac, neuronal, pancreatic, and hepatic lineage. The protocols include (1) the formation of embryoid bodies (EBs) followed by (2) the spontaneous differentiation of EBs into progenitor cells of the ecto-, endo-, and mesodermal germ layer and (3) the directed differentiation of early progenitors into the respective lineages. Differentiation induction via growth and extracellular matrix factors leads to titin-expressing spontaneously beating cardiac cells, tyrosine hydroxylase-expressing dopaminergic neurons, insulin and c-peptide co-expressing pancreatic islet-like clusters, and albumin-positive hepatic cells, respectively. The differentiated cells show tissue-specific proteins and electrophysiological properties (action potentials and ion channels) in cardiac and neuronal cells, glucose-dependent insulin release in pancreatic cells, or glycogen storage and albumin synthesis in hepatic cells. The protocols presented here provide basic systems to study differentiation processes in vitro and to establish strategies for the use of stem cells in regenerative therapies.

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Acknowledgments

We are grateful to Sabine Sommerfeld, Oda Weiss, and Karla Meier for technical assistance and Drs. Przemyslav Blyszczuk and Gabriela Kania for their expert help in the establishment of pancreatic and hepatic differentiation conditions. We are grateful to Dr. Dieter Fuerst, University of Bonn, Germany, for kindly providing antibodies. We thank the German Research Foundation (DFG) and the Ministry of Education and Research (BMBF) for funding our stem cell projects.

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  1. In Vitro Differentiation Group, Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

    Insa S. Schroeder, Cornelia Wiese, Thuy T. Truong, Alexandra Rolletschek & Anna M. Wobus

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  1. Insa S. Schroeder
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  2. Cornelia Wiese
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    Schroeder, I.S., Wiese, C., Truong, T.T., Rolletschek, A., Wobus, A.M. (2009). Differentiation Analysis of Pluripotent Mouse Embryonic Stem (ES) Cells In Vitro. In: Wurst, W., Kühn, R. (eds) Gene Knockout Protocols. Methods in Molecular Biology, vol 530. Humana Press. https://doi.org/10.1007/978-1-59745-471-1_12

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