title: Derivation of human embryonic stem cells using a post-inner cell mass intermediate.
abstract: Little is known about the true developmental origin of human embryonic stem cells (hESCs) or the events that initiate their generation. Recently, we have shown that hESCs originate from a post-inner cell mass (ICM) intermediate (PICMI), a unique transient epiblast-like structure that is different from both its ICM progenitor and its subsequent hESC fate. As a closer progenitor of hESCs than the ICM, the PICMI could be used to provide further insight into the human pluripotent state. Here we provide a detailed (7-d) protocol for the culture of the human preimplantation embryos in order to derive the PICMI. Subsequent identification and cryopreservation of the PICMI are described, in addition to hESC derivation. The initial hESC outgrowth is visible within 2-7 d after PICMI plating. By using the protocol provided, we observed PICMI formation in 21.3% of plated blastocysts with good-quality ICMs. Of the PICMIs used for hESC derivation, 80.6% showed hESC outgrowth after further culture.
authors.affiliation: Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium. toleary76@hotmail.com
authors.forename: Thomas
authors.initials: T
authors.lastname: O'Leary
authors.forename: Björn
authors.initials: B
authors.lastname: Heindryckx
authors.forename: Sylvie
authors.initials: S
authors.lastname: Lierman
authors.forename: Margot
authors.initials: M
authors.lastname: Van der Jeught
authors.forename: Galbha
authors.initials: G
authors.lastname: Duggal
authors.forename: Petra
authors.initials: P
authors.lastname: De Sutter
authors.forename: Susana M
authors.initials: SM
authors.lastname: Chuva de Sousa Lopes
mesh_terms: D053624:Blastocyst Inner Cell Mass / Q000166:cytology* / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D015925:Cryopreservation / Q000379:methods*; D046149:Embryo Culture Techniques / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology*; D005855:Germ Layers / Q000166:cytology* / Q000196:embryology; D006801:Humans