title: Cyclin E1 plays a key role in balancing between totipotency and differentiation in human embryonic cells.
abstract: OBJECTIVE
  We aimed to investigate if Cyclin E1 (CCNE1) plays a role in human embryogenesis, in particular during the early developmental stages characterized by a short cell cycle.
  
  
  RESULTS
  CCNE1 is expressed in plenipotent human embryonic cells and plays a critical role during hESC derivation via the naïve state and, potentially, normal embryo development.
  
  
  BACKGROUND
  A short cell cycle due to a truncated G1 phase has been associated with the high developmental capacity of embryonic cells. CCNE1 is a critical G1/S transition regulator. CCNE1 overexpression can cause shortening of the cell cycle and it is constitutively expressed in mouse embryonic stem cells and cancer cells.
  
  
  METHODS
  We investigated expression of CCNE1 in human preimplantation embryo development and embryonic stem cells (hESC). Functional studies included CCNE1 overexpression in hESC and CCNE1 downregulation in the outgrowths formed by plated human blastocysts. Analysis was performed by immunocytochemistry and quantitative real-time PCR. Mann-Whitney statistical test was applied.
  
  
  RESULTS
  The CCNE1 protein was ubiquitously and constitutively expressed in the plenipotent cells of the embryo from the 4-cell stage up to and including the full blastocyst. During blastocyst expansion, CCNE1 was downregulated in the trophectoderm (TE) cells. CCNE1 shortly co-localized with NANOG in the inner cell mass (ICM) of expanding blastocysts, mimicking the situation in naïve hESC. In the ICM of expanded blastocysts, which corresponds with primed hESC, CCNE1 defined a subpopulation of cells different from NANOG/POU5F1-expressing pluripotent epiblast (EPI) cells and GATA4/SOX17-expressing primitive endoderm (PrE) cells. This CCNE1-positive cell population was associated with visceral endoderm based on transthyretin expression and marked the third cell lineage within the ICM, besides EPI and PrE, which had never been described before. We also investigated the role of CCNE1 by plating expanded blastocysts for hESC derivation. As a result, all the cells including TE cells re-gained CCNE1 and, consequently, NANOG expression, resembling the phenotype of naïve hESC. The inhibition of CCNE1 expression with siRNA blocked proliferation and caused degeneration of those plated cells.
  
  
  CONCLUSIONS
  The study is based on a limited number of good-quality human embryos donated to research.
  
  
  CONCLUSIONS
  Our study sheds light on the processes underlying the high developmental potential of early human embryonic cells. The CCNE1-positive plenipotent cell type corresponds with a phenotype that enables early human embryos to recover after fragmentation, cryodamage or (single cell) biopsy on day 3 for preimplantation genetic diagnosis. Knowledge on the expression and function of genes responsible for this flexibility will help us to better understand the undifferentiated state in stem cell biology and might enable us to improve technologies in assisted reproduction.
  
  
  METHODS
  NA STUDY FUNDING AND COMPETING INTERESTS: This research is supported by grants from the Fund for Scientific Research - Flanders (FWO-Vlaanderen), the Methusalem (METH) of the VUB and Scientific Research Fond Willy Gepts of UZ Brussel. There are no competing interests.
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium krivega@gmail.com.
authors.forename: M V
authors.initials: MV
authors.lastname: Krivega
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Geens
authors.affiliation: Ghent Fertility and Stem Cell Team, Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium.
authors.forename: B
authors.initials: B
authors.lastname: Heindryckx
authors.affiliation: Centre for Reproductive Medicine (CRG), Brussels University Hospital, Laarbeeklaan 101, 1090 Brussels, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Santos-Ribeiro
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium Centre for Reproductive Medicine (CRG), Brussels University Hospital, Laarbeeklaan 101, 1090 Brussels, Belgium.
authors.forename: H
authors.initials: H
authors.lastname: Tournaye
authors.affiliation: Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium Centre for Reproductive Medicine (CRG), Brussels University Hospital, Laarbeeklaan 101, 1090 Brussels, Belgium.
authors.forename: H
authors.initials: H
authors.lastname: Van de Velde
mesh_terms: D017209:Apoptosis / Q000502:physiology; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019700:Thrombospondin 1 / Q000378:metabolism; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
chemical_list: C475820:CCNE1 protein, human; D019927:Cyclin E; D015513:Oncogene Proteins; D019700:Thrombospondin 1; D014660:Vasoactive Intestinal Peptide
keywords: cell cycle; cell proliferation; cyclin E1; embryo development; human embryo; naive hESC; plenipotency; stem cells; totipotency; visceral endoderm