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

The optimization of human embryonic stem (hES) cell line derivation methods is challenging be-cause many worldwide laboratories have neither access to spare human embryos nor ethical ap-proval for using supernumerary human embryos for hES cell derivation purposes. Additionally, studies performed directly on human embryos imply a waste of precious human biological mater-ial. In this study, we developed a new strategy based on the combination of whole-blastocyst cul-ture followed by laser drilling destruction of the trophoectoderm for improving the efficiency of in-ner 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 ap-plied to poor-quality blastocyts that otherwise would not be suitable for laser drilling itself nor im-munosurgery 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 typica