TY - CHAP

T1 - Human Embryonic and Hepatic Stem Cell Differentiation Visualized in Two and Three Dimensions Based on Serial Sections

AU - Vestentoft, Peter S.

AU - Brøchner, Christian B

AU - Lynnerup, Niels

AU - Andersen, Claus Yding

AU - Møllgård, Kjeld

PY - 2015

Y1 - 2015

N2 - Pluripotent human embryonic stem cells (hESCs) are characterized by two defining properties, self-renewal and differentiation. Self-renewing hESCs express transcription factors OCT4, SOX2, and NANOG, and surface markers SSEA-4 and TRA-1-60 and TRA-1-81 and their ability to differentiate into derivatives of the three germ layers show the differentiating potential. Studies suggest a certain microheterogeneity of the hESC colonies, in which not all cells in one colony of apparently undifferentiated cells express all the expected markers. We describe a technique to paraffin embed an entire hESC colony, and prepare 3-5 μm thick serial sections. Immunohistochemistry applied to individual sections produces a 2-dimensional survey of the developing hESC colony. Based on serial paraffin sections of the 2D-expression pattern, a new and useful 3D-visualization can be modeled. The actual 3D rendering of an entire colony is accomplished using 3D image processing software such as Mimics(®) or Amira(®). An extended version of this technique even allows for a high-magnification 3D-reconstruction of, e.g., hepatic stem cells in developing liver. These techniques combined allow for both a 2- and a 3-dimensional visualization of hESC colonies and stem cells in organs, which leads to new insights into and information about the interaction of stem cells with their surroundings.

AB - Pluripotent human embryonic stem cells (hESCs) are characterized by two defining properties, self-renewal and differentiation. Self-renewing hESCs express transcription factors OCT4, SOX2, and NANOG, and surface markers SSEA-4 and TRA-1-60 and TRA-1-81 and their ability to differentiate into derivatives of the three germ layers show the differentiating potential. Studies suggest a certain microheterogeneity of the hESC colonies, in which not all cells in one colony of apparently undifferentiated cells express all the expected markers. We describe a technique to paraffin embed an entire hESC colony, and prepare 3-5 μm thick serial sections. Immunohistochemistry applied to individual sections produces a 2-dimensional survey of the developing hESC colony. Based on serial paraffin sections of the 2D-expression pattern, a new and useful 3D-visualization can be modeled. The actual 3D rendering of an entire colony is accomplished using 3D image processing software such as Mimics(®) or Amira(®). An extended version of this technique even allows for a high-magnification 3D-reconstruction of, e.g., hepatic stem cells in developing liver. These techniques combined allow for both a 2- and a 3-dimensional visualization of hESC colonies and stem cells in organs, which leads to new insights into and information about the interaction of stem cells with their surroundings.

U2 - 10.1007/7651_2014_128

DO - 10.1007/7651_2014_128

M3 - Book chapter

C2 - 25304207

SN - 978-1-4939-2667-1

VL - 1307

T3 - Methods in Molecular Biology

SP - 245

EP - 262

BT - Human Embryonic Stem Cell Protocols

PB - Springer Science+Business Media

ER -