TY - JOUR

T1 - A two- and three-dimensional approach for visualizing human embryonic stem cell differentiation

T2 - chapter 10

AU - Brøchner, Christian Beltoft

AU - Vestentoft, Peter S

AU - Lynnerup, Niels

AU - Andersen, Claus Yding

AU - Møllgård, Kjeld

PY - 2010

Y1 - 2010

N2 - 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.

AB - 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.

U2 - 10.1007/978-1-60761-369-5_10

DO - 10.1007/978-1-60761-369-5_10

M3 - Journal article

C2 - 19907978

VL - 584

SP - 179

EP - 193

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -