Abstract
Pre-implantation genetic diagnosis allows the characterisation of embryos that carry a gene responsible for a severe monogenic disease and to transfer to the mother’s uterus only the unaffected one(s). The genetically affected embryos can be used to establish human embryonic stem cell (hESC) lines. We are currently establishing a cell bank of ESC lines carrying specific disease-causing mutant genes. These cell lines are available to the scientific community. For this purpose, we have designed a technique that requires only minimal manipulation of the embryos. At the blastocyst stage, we just removed the zona pellucida before seeding the embryo as a whole on a layer of feeder cells. This approach gave a good success rate (>20%), whatever the quality of the embryos, and allowed us to derive 11 new hESC lines, representing seven different pathologies. Full phenotypic validation of the cell lines according to ISCI guidelines confirmed their pluripotent nature, as they were positive for hESC markers and able to differentiate in vitro in all three germ layers derivatives. Nine out of 11 stem cell lines had normal karyotypes. Our results indicate that inner cell mass isolation is not mandatory for hESC derivation and that minimal manipulation of embryos can lead to high success rate.
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Acknowledgements
We would like to thank Nicolas Becker, Nathalie Gardes and Jean Christophe Nicod for their technical help and the IVF team at the SIHCUS-CMCO. We also would like to thank Bruno Costes for his precious help with the STR genotyping and Xavier Nissan for the TLDA analysis. This work was supported by grants from the Association Française contre les Myopathies (AFM), the Agence Nationale pour la Recherche (ANR, hESCREEN), Medicen Paris Region (IngeCell network), the Centre National de la Recherche Scientifique (CNRS) and the Institut National de la Santé et de la Recherche Médicale (INSERM).
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Editor: P. Andrews
P. Tropel and J. Tournois contributed equally to this study.
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Tropel, P., Tournois, J., Côme, J. et al. High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis. In Vitro Cell.Dev.Biol.-Animal 46, 376–385 (2010). https://doi.org/10.1007/s11626-010-9300-8
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DOI: https://doi.org/10.1007/s11626-010-9300-8