title: Utilizing FUCCI reporters to understand pluripotent stem cell biology.
abstract: The fluorescence ubiquitination cell cycle indicator (FUCCI) system provides a powerful method to evaluate cell cycle mechanisms associated with stem cell self-renewal and cell fate specification. By integrating the FUCCI system into human pluripotent stem cells (hPSCs) it is possible to isolate homogeneous fractions of viable cells representative of all cell cycle phases. This method avoids problems associated with traditional tools used for cell cycle analysis such as synchronizing drugs, elutriation and temperature sensitive mutants. Importantly, FUCCI reporters allow cell cycle events in dynamic systems, such as differentiation, to be evaluated. Initial reports on the FUCCI system focused on its strengths in reporting spatio-temporal aspects of cell cycle events in living cells and developmental models. In this report, we describe approaches that broaden the application of FUCCI reporters in PSCs through incorporation of FACS. This approach allows molecular analysis of the cell cycle in stem cell systems that were not previously possible.
authors.affiliation: Department of Biochemistry and Molecular Biology, Paul D. Coverdell Center for Biomedical and Health Sciences, The University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA.
authors.forename: Amar M
authors.initials: AM
authors.lastname: Singh
authors.affiliation: Department of Biochemistry and Molecular Biology, Paul D. Coverdell Center for Biomedical and Health Sciences, The University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Trost
authors.affiliation: Department of Biochemistry and Molecular Biology, Paul D. Coverdell Center for Biomedical and Health Sciences, The University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Boward
authors.affiliation: Department of Biochemistry and Molecular Biology, Paul D. Coverdell Center for Biomedical and Health Sciences, The University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA. Electronic address: sdalton@uga.edu.
authors.forename: Stephen
authors.initials: S
authors.lastname: Dalton
mesh_terms: D018929:Cell Culture Techniques; D002453:Cell Cycle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D039904:Pluripotent Stem Cells / Q000502:physiology*; D054875:Ubiquitination; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: Cell cycle; Differentiation; Epigenetics; Pluripotent stem cells