A two- and three-dimensional approach for visualizing human embryonic stem cell differentiation.
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.
Brøchner
Christian B
CB
Department of Cellular and Molecular Medicine, University of Copenhagen, The Panum Institute, Copenhagen N, Denmark.
Vestentoft
Peter S
PS
Lynnerup
Niels
N
Andersen
Claus Yding
CY
Møllgård
Kjeld
K
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
D015415:Biomarkers