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(620) NUCLEAR TRANSFER REPROGRAMMING DOES NOT IMPROVE THE LOW DEVELOPMENTAL POTENCY OF EMBRYONIC STEM CELL WITH EXTENSIVE PASSAGES. Amano, Tomokazu1, Kurihara, Yukiko2, Uchijima, Yasunobu2, Kurihara, Hiroki2, Suzuki, Hiroshi1, 3, 1 Department of Developmental and Medical technology, Tokyo, Japan2 Department of Physiological and Metabolism, Tokyo, Japan3 National Research Center for Protozoan Diseases, Obihiro, Japan ABSTRACT- Cloned mice have been produced after nuclear transfer of ES cells. However, majority of the pups obtained after transfer of cloned embryos has died soon after birth indicating that especially ES cells already had strictly epigenetic modification during in vitro culture. To compare the ability of ES cell line with extensive passages and nt-ES cells derived from it, in vitro differentiation ability and developmental potential to term was examined by suspension culture and tetraploid complementation in this study. B6C3HF1 mice were used as oocyte donors throughout. Nuclear transfer cloning of TT2 ES cell with extensive passages (ep-ES) was performed by electrofusion method. The oocytes fused with ES cell were cultured in KSOM medium for 1h before activation. After activation by 10 mM Sr2+, these embryos were further cultured to blastocyst stage to establish nt-ES cell line. To evaluate in vitro differentiation ability, both ep- and nt-ES cells were induced to differentiate into simple (SEB) and cystic (CEB) embryoid body in suspension culture. Moreover, for developmental potential in vivo, these ES cells were aggregated with tetraploid embryos and efficiency to term was observed after embryo transfer. In ep-ES cells, appearance for SEB, CEB and beating was 3, 6 and 9 days after cultivation, respectively. In nt-ES cell lines tested, appearance for SEB and CEB were almost similar (3 days for SEB, 6 to 9 days for CEB). However, no beating was observed within 3 weeks for one of nt-ES cell lines indicating that there was a difference regarding in vitro differentiation ability between nt- and ep-ES cell. The developmental rate of ep-ES cells aggregated with tetraploid embryos to term was 6%. But majority of live births (80%) were died soon after birth. In the nt-ES cell lines, production efficiency of extensively ES-derived mice after tetraploid aggregation was ranging from 0 to 5%. Moreover, those pups were also died soon after birth similarly ep-ES derived pups. Therefore, developmental potential to term or survival rates after birth was not recovered even if they were with several passages. These results suggested that epigenetic alterations of ES cell were retained at cell line re-established by nuclear transfer. KEY WORDS: reprogramming, tetraploid complementation, nuclear transfer, ES cell |
