title: Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos.
abstract: BACKGROUND
  Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres.
  
  
  METHODS
  Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs).
  
  
  RESULTS
  Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT-PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype.
  
  
  CONCLUSIONS
  We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.
authors.affiliation: Department of Embryology and Genetics, Vrije Universiteit Brussel, 1090 Brussels, Belgium.
authors.forename: Mieke
authors.initials: M
authors.lastname: Geens
authors.forename: Ileana
authors.initials: I
authors.lastname: Mateizel
authors.forename: Karen
authors.initials: K
authors.lastname: Sermon
authors.forename: Martine
authors.initials: M
authors.lastname: De Rycke
authors.forename: Claudia
authors.initials: C
authors.lastname: Spits
authors.forename: Greet
authors.initials: G
authors.lastname: Cauffman
authors.forename: Paul
authors.initials: P
authors.lastname: Devroey
authors.forename: Herman
authors.initials: H
authors.lastname: Tournaye
authors.forename: Inge
authors.initials: I
authors.lastname: Liebaers
authors.forename: Hilde
authors.initials: H
authors.lastname: Van de Velde
mesh_terms: D001757:Blastomeres / Q000166:cytology*; D002460:Cell Line; D046149:Embryo Culture Techniques; D047108:Embryonic Development; D053595:Embryonic Stem Cells; D006801:Humans; D039904:Pluripotent Stem Cells