title: A two- and three-dimensional approach for visualizing human embryonic stem cell differentiation.
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 microm thick) and prepare 2-microm 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.
authors.affiliation: Department of Cellular and Molecular Medicine, University of Copenhagen, The Panum Institute, Copenhagen N, Denmark.
authors.forename: Christian B
authors.initials: CB
authors.lastname: Brøchner
authors.forename: Peter S
authors.initials: PS
authors.lastname: Vestentoft
authors.forename: Niels
authors.initials: N
authors.lastname: Lynnerup
authors.forename: Claus Yding
authors.initials: CY
authors.lastname: Andersen
authors.forename: Kjeld
authors.initials: K
authors.lastname: Møllgård
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods* / Q000706:statistics & numerical data; D007150:Immunohistochemistry; D016612:Paraffin Embedding / Q000379:methods; D012984:Software; D013194:Staining and Labeling
chemical_list: D015415:Biomarkers