PURPOSES: Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free 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 xeno-free culture conditions. METHODS: Cumulus-oocyte-complexes were in vitro matured and activated using Ca(2+)Ionophore and 6-DMAP 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 Oct-4. RESULTS: Parthenogenesis 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 Oct-4 and they all tested positive for this pluripotency marker. CONCLUSION: Our data show that Ca(2+) Ionophore, and 6-DMAP 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 Stem-Pro 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.
PURPOSES: Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free 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 xeno-free culture conditions. METHODS: Cumulus-oocyte-complexes were in vitro matured and activated using Ca(2+)Ionophore and 6-DMAP 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 Oct-4. RESULTS: Parthenogenesis 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 Oct-4 and they all tested positive for this pluripotency marker. CONCLUSION: Our data show that Ca(2+) Ionophore, and 6-DMAP 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 Stem-Pro 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.
Authors: Catharina Ellerström; Raimund Strehl; Karina Moya; Katarina Andersson; Christina Bergh; Kersti Lundin; Johan Hyllner; Henrik Semb Journal: Stem Cells Date: 2006-06-01 Impact factor: 6.277
Authors: R M Ferreira; H Ayres; M R Chiaratti; M L Ferraz; A B Araújo; C A Rodrigues; Y F Watanabe; A A Vireque; D C Joaquim; L C Smith; F V Meirelles; P S Baruselli Journal: J Dairy Sci Date: 2011-05 Impact factor: 4.034
Authors: J A Thomson; J Itskovitz-Eldor; S S Shapiro; M A Waknitz; J J Swiergiel; V S Marshall; J M Jones Journal: Science Date: 1998-11-06 Impact factor: 47.728
Authors: Adriana Bos-Mikich; Fabiana F Bressan; Rafael R Ruggeri; Yeda Watanabe; Flávio V Meirelles Journal: Stem Cells Int Date: 2015-11-09 Impact factor: 5.443