A. Rybouchkin, Y. Kato, Y. Tsunoda

One of the limiting factors in production of the human somatic cell nuclear transfer embryonic stem (ntES) cell lines is the paucity of the donor oocyte source and furthermore rather ineffective use of these oocytes for production of NT blastocysts (starting material for ES cell derivation). Indeed, while currently available culture media allow obtaining about 60-70% of blastocysts from in vitro fertilized human oocytes, at present only 25-30% of NT blastocysts obtained after transfer of fibroblast or cumulus cell nuclei can develop to blastocyst stage. The likely reason for reduced development of the NT embryos to the blastocysts stage is an imperfect epigenetic reprogramming of the somatic cell derived chromatin. We have studied the process of epigenetic reprogramming in the mouse model of somatic cell nuclear transfer by applying histone deacetylation inhibitor trichostatin A (TSA).

The freshly ovulated mouse cumulus-oocyte complexes (COCs) were recovered from B6D2F1 hybrid females at 13-14 hrs following hormonally induced superovulation. The COCs were treated with hyaluronidase to remove cumulus cells, which were later used as nuclear donors. Oocytes were enucleated with 7μm i.d. micropipette under control of piezo-driven manipulator set (PMAS-CT150/MMO202N) in the FHM medium supplemented with 5μg/ml cytochalasin B. After enucleation they were injected in plain FHM medium with cumulus cell nuclei using the same manipulator set and smaller (5μm i.d.) micropipette. After injection they were split onto two groups (A and B). Group A was incubated in plain KSOM medium, while group B in KSOM supplemented with 100nM TSA. Following 2-3 hrs of incubation NT oocytes were activated by incubation for 6 hrs in Ca2+free KSOM supplemented with 10 mM SrCl2 and 5μg/ml cytochalasin B. Additionally, for group B activation medium was supplemented with 100nM TSA. Following activation, embryos were washed through several drops of KSOM and cultured further for 90-92 hrs in KSOM. At the end of culture the numbers of blastocysts were estimated. The quality of the blastocysts was also analyzed by differential staining and cell number counting. In total after three independent repeats statistically significantly more embryos reach blastocysts stage in group B than in group A (67% of 98 vs 39% of 96, recpectively, p

We concluded that the treatment of NT oocytes before and during activation period with TSA leads to the significant increase in the number of embryos reaching blastocyst stage. Moreover, more blastocyst has high cell number, especially in the ICM, which is particularly important for successful derivation of ES cell lines. This approach would be very tempting to verify in the human.

This work was supported by the grant from PROBRAIN.

Keywords (Optional): 
therapeutic cloning
nuclear transfer
embryonic stem cells