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The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Ruggeri, R. R. ; Watanabe, Y. ; Meirelles, F. ; Bressan, F. F. ; Frantz, N. ; Bos-Mikich, A.

Journal of assisted reproduction and genetics, 2012-10, Vol.29 (10), p.1039-1043 [Periódico revisado por pares]

Boston: Springer US

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Título:
The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions
Autor: Ruggeri, R. R. ; Watanabe, Y. ; Meirelles, F. ; Bressan, F. F. ; Frantz, N. ; Bos-Mikich, A.
Assuntos: Adenine - analogs & derivatives ; Adenine - pharmacology ; Alkaline Phosphatase - metabolism ; Animals ; Biomarkers - analysis ; Biomarkers - metabolism ; Blastocyst - cytology ; Blastocyst - drug effects ; Blastocyst - physiology ; Blastocyst Inner Cell Mass - cytology ; Cattle ; Embryo Culture Techniques ; Embryonic Stem Cells - cytology ; Embryos ; Female ; Fertilization in Vitro ; Fibronectins - chemistry ; Gynecology ; Human Genetics ; In vitro fertilization ; Male ; Medicine ; Medicine & Public Health ; Octamer Transcription Factor-3 - metabolism ; Parthenogenesis ; Phosphatase ; Pluripotent Stem Cells - metabolism ; Proprietary ; Reproductive Medicine ; Stem Cell Biology ; Stem cells
É parte de: Journal of assisted reproduction and genetics, 2012-10, Vol.29 (10), p.1039-1043
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Descrição: 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.
Editor: Boston: Springer US
Idioma: Inglês

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The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Ruggeri, R. R. ; Watanabe, Y. ; Meirelles, F. ; Bressan, F. F. ; Frantz, N. ; Bos-Mikich, A.

Boston: Springer US

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