Phenotypic analysis of human embryonic stem cells.
Human embryonic stem cells (hESCs) are an important tool for the study of developmental biology and may one day serve as a source of cells for regenerative medicine. As no definitive assay for hESC pluripotency is available, surrogate assays that measure markers or properties that have been correlated with hESC developmental potential are used to measure the effects of test conditions on their propagation and differentiation. This unit presents a range of protocols, including visual inspection, flow cytometry, immunofluorescence, quantitative real-time reverse-transcriptase PCR, and a colony-forming assay, as tools to measure the undifferentiated hESC state. The authors discuss the advantages and limitations of the various protocols, and present expected results and discuss potential problems. The development of quantitative assays of hESC developmental potential are critical for our understanding of hESC biology.
Ungrin
Mark
M
IBBME, University of Toronto, Toronto, Canada.
O'Connor
Michael
M
Eaves
Connie
C
Zandstra
Peter W
PW
D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D003114:Colony-Forming Units Assay; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers