Authors: R R Ruggeri Y Watanabe F Meirelles F F Bressan N Frantz A BosMikich
Publish Date: 2012/09/29
Volume: 29, Issue: 10, Pages: 1039-1043
Abstract
Clinical application of human embryonic stem cells will be possible when cell lines are created under xenofree and defined conditions We aimed to establish methodologies for parthenogenetic activation culture to blastocyst and mechanical isolation of the inner cell mass ICM using bovine oocytes as a model for derivation and proliferation of human embryonic stem cells under defined xenofree culture conditionsCumulusoocytecomplexes were in vitro matured and activated using Ca2+Ionophore and 6DMAP or in vitro fertilized IVF Parthenotes and biparental embryos were cultured to blastocysts when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemPro® medium After attachment primary colonies were left to proliferate and stained for pluripotency markers alkaline phosphatase and Oct4Parthenogenesis and fertilization presented significantly different success rates 91 and 79 respectively and blastocyst formation 40 and 43 respectively ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates 39 and 33 respectively Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase Three colonies were analyzed for Oct4 and they all tested positive for this pluripotency markerOur data show that Ca2+ Ionophore and 6DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development After mechanical isolation parthenogetic derived ICMs showed a good rate of derivation in fibronectin and StemPro forming primary and expanded colonies of putative embryonic stem cells This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions
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