title: Whole-blastocyst culture followed by laser drilling technology enhances the efficiency of inner cell mass isolation and embryonic stem cell derivation from good- and poor-quality mouse embryos: new insights for derivation of human embryonic stem cell lines.
abstract: The optimization of human embryonic stem (hES) cell line derivation methods is challenging because many worldwide laboratories have neither access to spare human embryos nor ethical approval for using supernumerary human embryos for hES cell derivation purposes. Additionally, studies performed directly on human embryos imply a waste of precious human biological material. In this study, we developed a new strategy based on the combination of whole-blastocyst culture followed by laser drilling destruction of the trophoectoderm for improving the efficiency of inner cell mass (ICM) isolation and ES cell derivation using murine embryos. Embryos were divided into good- and poor-quality embryos. We demonstrate that the efficiency of both ICM isolation and ES cell derivation using this strategy is significantly superior to whole-blastocyst culture or laser drilling technology itself. Regardless of the ICM isolation method, the ES cell establishment depends on a feeder cell growth surface. Importantly, this combined methodology can be successfully applied to poor-quality blastocysts that otherwise would not be suitable for laser drilling itself nor immunosurgery in an attempt to derive ES cell lines due to the inability to distinguish the ICM. The ES cell lines derived by this combined method were characterized and shown to maintain a typical morphology, undifferentiated phenotype, and in vitro and in vivo three germ layer differentiation potential. Finally, all ES cell lines established using either technology acquired an aneuploid karyotype after extended culture periods, suggesting that the method used for ES cell derivation does not seem to influence the karyotype of the ES cells after extended culture. This methodology may open up new avenues for further improvements for the derivation of hES cells, the majority of which are derived from frozen, poor-quality human embryos.
authors.affiliation: Spanish Stem Cell Bank (Andalusian Branch), University of Granada, Instituto de Investigaciones Biomédicas, Parque Tecnológico de la Salud, Avda del Conocimiento s/n, Granada, Spain. josel.cortes.sspa@juntadeandalucia.es
authors.forename: J L
authors.initials: JL
authors.lastname: Cortes
authors.forename: L
authors.initials: L
authors.lastname: Sánchez
authors.forename: P
authors.initials: P
authors.lastname: Catalina
authors.forename: F
authors.initials: F
authors.lastname: Cobo
authors.forename: C
authors.initials: C
authors.lastname: Bueno
authors.forename: A
authors.initials: A
authors.lastname: Martínez-Ramirez
authors.forename: A
authors.initials: A
authors.lastname: Barroso
authors.forename: C
authors.initials: C
authors.lastname: Cabrera
authors.forename: G
authors.initials: G
authors.lastname: Ligero
authors.forename: R
authors.initials: R
authors.lastname: Montes
authors.forename: R
authors.initials: R
authors.lastname: Rubio
authors.forename: A
authors.initials: A
authors.lastname: Nieto
authors.forename: P
authors.initials: P
authors.lastname: Menendez
mesh_terms: D000465:Algorithms; D000818:Animals; D053624:Blastocyst Inner Cell Mass / Q000166:cytology* / Q000502:physiology; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D046149:Embryo Culture Techniques / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology; D005260:Female; D006801:Humans; D007621:Karyotyping; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D011247:Pregnancy; D011786:Quality Control