High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis.
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.
Tropel
Philippe
P
IGBMC, Illkirch 67404, France.
Tournois
Johana
J
Côme
Julien
J
Varela
Christine
C
Moutou
Céline
C
Fragner
Pascal
P
Cailleret
Michel
M
Laâbi
Yacine
Y
Peschanski
Marc
M
Viville
Stéphane
S
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001755:Blastocyst / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007621:Karyotyping; D008297:Male; D051379:Mice; D010375:Pedigree; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D019836:Preimplantation Diagnosis / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000954:Antigens, Surface; D015415:Biomarkers