title: Lengthened G1 phase indicates differentiation status in human embryonic stem cells.
abstract: The cell cycle in pluripotent stem cells is notable for the brevity of the G1 phase, permitting rapid proliferation and reducing the duration of differentiation signal sensitivity associated with the G1 phase. Changes in the length of G1 phase are understood to accompany the differentiation of human embryonic stem cells (hESCs), but the timing and extent of such changes are poorly defined. Understanding the early steps governing the differentiation of hESCs will facilitate better control over differentiation for regenerative medicine and drug discovery applications. Here we report the first use of real-time cell cycle reporters in hESCs. We coexpressed the chromatin-decorating H2B-GFP fusion protein and the fluorescence ubiquitination cell cycle indicator (FUCCI)-G1 fusion protein, a G1 phase-specific reporter, in hESCs to measure the cell cycle status in live cells. We found that FUCCI-G1 expression is weakly detected in undifferentiated hESCs, but rapidly increases upon differentiation. hESCs in the G1 phase display a reduction in undifferentiated colony-initiating cell function, underscoring the relationship between G1 phase residence and differentiation. Importantly, we demonstrate inter- and intracolony variation in response to chemicals that induce differentiation, implying extensive cell-cell variation in the threshold necessary to alter the G1 phase length. Finally, gain of differentiation markers appears to be coincident with G1 phase lengthening, with distinct G1 phase profiles associated with different markers of early hESC differentiation. Our data demonstrate the tight coupling of cell cycle changes to hESC differentiation, and highlight the cell cycle reporter system and assays we have implemented as a novel avenue for investigating pluripotency and differentiation.
authors: McMaster Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, McMaster University , Hamilton, Ontario, Canada.
authors: Ashley
authors: A
authors: Calder
authors: Ivana
authors: I
authors: Roth-Albin
authors: Sonam
authors: S
authors: Bhatia
authors: Carlos
authors: C
authors: Pilquil
authors: Jong Hee
authors: JH
authors: Lee
authors: Mick
authors: M
authors: Bhatia
authors: Marilyne
authors: M
authors: Levadoux-Martin
authors: Jamie
authors: J
authors: McNicol
authors: Jennifer
authors: J
authors: Russell
authors: Tony
authors: T
authors: Collins
authors: Jonathan S
authors: JS
authors: Draper
mesh_terms: D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002465:Cell Movement; D049109:Cell Proliferation; D003470:Culture Media / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D016193:G1 Phase; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D059008:Time-Lapse Imaging; D019076:Transgenes
chemical_list: D000943:Antigens, Differentiation; D015415:Biomarkers; D003470:Culture Media; D050991:GATA4 Transcription Factor; C484170:GATA4 protein, human; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins