Abstract
Undifferentiated human embryonic stem cells are characterized by expression of specific cell markers like the transcription factors OCT4, SOX2, and NANOG, the stage-specific embryonic antigen SSEA4, and the tumor-related antigens TRA-1-60 and TRA-1-81 and by their ability to differentiate under proper conditions into cells of the three germ layers and later into derivatives of these germ layers. Recent studies suggest a certain micro-heterogeneity of the expression of hESC markers, which demonstrates that not all cells in a hESC colony of apparently undifferentiated cells express all the expected markers. We describe a technique allowing paraffin embedding an entire hESC colony (e.g., ˜ 150 µm thick) and prepare 2-µm thick serial sections. Different staining procedures applied to individual sections produce a 2D survey of the developing hESC colony. Furthermore, a new and useful visualization of this 2D-expression pattern can be created by developing a 3D-model of the culture, based on serial paraffin sections. Individual sections are stained using individual markers. Using 3D image processing software such as Mimics or 3D-Doctor, the actual 3D-rendering of an entire colony can be accomplished. An extended version of this technique even allows for a high-magnification 3D-reconstruction of an area of interest (AOI), e.g., the developing hepatic stem cells. These techniques allow both a 2D and a 3D visualization of hESC colonies and lead to new insights into and information about the interaction of stem cells.
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Acknowledgments
The authors would like to thank Sussi Forchhammer, Hanne Hadberg, Pernille Froh, and Ha Nguyen for the expert technical assistance. Keld B. Ottesen for the final layout of the photos.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC 2006
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Brøchner, C.B., Vestentoft, P.S., Lynnerup, N., Andersen, C.Y., Møllgård, K. (2009). A Two- and Three-Dimensional Approach for Visualizing Human Embryonic Stem Cell Differentiation. 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_10
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DOI: https://doi.org/10.1007/978-1-60761-369-5_10
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