213 WNT inhibition by dickkopf WNT signalling pathway inhibitor 1 (DKK1) cannot replace IWR-1 during derivation of bovine embryonic stem cells

Understanding the signalling pathways involved with derivation of embryonic stem cells could enhance our understanding of pluripotency in pre-implantation embryos. Recently, the small molecule IWR-1 has been shown to promote derivation of mouse epiblast stem cells and pluripotent bovine and porcine embryonic stem cells (ESC). IWR-1 blocks WNT signalling mediated by β-catenin-targeted gene expression through stabilisation of Axin2, a member of the destruction complex that induces β-catenin degradation. Here, we evaluated whether dickkopf WNT signalling pathway inhibitor 1 (DKK1) can replace IWR-1 for establishment of bovine pluripotent ESC. If so, it is likely that the actions of IWR-1 to promote pluripotency involve inhibition of WNT signalling. Treatment of bovine embryos with 100ngmL−1 recombinant human DKK1 beginning at Day 5 of development decreased (P=0.02) immunofluorescent labelling of β-catenin in the resulting blastocysts (n=41-45/group), indicating that bovine embryos are responsive to DKK1 treatment. For ESC derivation, blastocysts were plated on top of feeder cells and cultured in ESC medium supplemented with 2.5 µM IWR-1 (n=21), 100ngmL−1 DKK1 (n=34), or vehicle (n=23). Cells were passaged every 5 to 7 days in their respective treatment medium. Seven days after plating, 57.9±14.7% of blastocysts in IWR-1 ESC medium developed outgrowth, which was lower (P=0.02) than the proportion of blastocysts with outgrowth in DKK1 medium (92.4±5.2%) or vehicle (81.9±10.0%). Outgrowth size did not differ among treatments. Labelling with CDX2 indicated that the majority of cells in outgrowths were trophectoderm cells. Thus, IWR-1 inhibits competence of blastocysts to form trophectoderm outgrowths during derivation of ESC. The percent of blastocysts from which cell lines were derived after 4 passages were 48% (10/21) for IWR-1, 41% (14/34) for DKK1, and 48% (11/23) for vehicle. Immunolabelling for the pluripotency marker SOX2 showed that only cells grown in IWR-1 medium were positive, whereas most of the cells derived in the other two media were not. Thus, IWR-1 could not be replaced by DKK1 for maintaining pluripotency. Immunoreactive β-catenin was abundantly distributed on the membrane of cells cultured with IWR-1 but not with DKK1 or vehicle-treated cells. Thus, β-catenin distribution to the cell membrane is linked with bovine pluripotency. Overall, results indicate that maintenance of pluripotency by IWR-1 may involve mechanisms other than WNT inhibition, and may be related to the localization of β-catenin to the plasma membrane.

Morphokinetics of cloned mouse embryos treated with epigenetic drugs and blastocyst prediction

Time-lapse monitoring of somatic cell nuclear transfer (SCNT) embryos may help to predict developmental success and increase birth and embryonic stem cells (ESC) derivation rates. Here, the development of ICSI fertilized embryos and of SCNT embryos, non-treated or treated with either psammaplin A (PsA) or vitamin C (VitC), was monitored, and the ESC derivation rates from the resulting blastocysts were determined. Blastocyst rates were similar among PsA-treated and VitC-treated SCNT embryos and ICSI embryos, but lower for non-treated SCNT embryos. ESC derivation rates were higher in treated SCNT embryos than in non-treated or ICSI embryos. Time-lapse microscopy analysis showed that non-treated SCNT embryos had a delayed development from the second division until compaction, lower number of blastomeres at compaction and longer compaction and cavitation durations compared with ICSI ones. Treatment of SCNT embryos with PsA further increased this delay whereas treatment with VitC slightly reduced it, suggesting that both treatments act through different mechanisms, not necessarily related to their epigenetic effects. Despite these differences, the time of completion of the third division, alone or combined with the duration of compaction and/or the presence of fragmentation, had a strong predictive value for blastocyst formation in all groups. In contrast, we failed to predict ESC derivation success from embryo morphokinetics. Time-lapse technology allows the selection of SCNT embryos with higher developmental potential and could help to increase cloning outcomes. Nonetheless, further studies are needed to find reliable markers for full-term development and ESC derivation success.

289 DERIVATION OF MOUSE EMBRYONIC STEM CELL FROM C57BL/6/EGFP STRAIN WITH FETAL CALF SERUM AND KNOCKOUT SERUM REPLACEMENT® SUPPLEMENTATION MEDIUM

Embryonic stem cells (ESC) have been used in attempts to obtain specific tissues or even individuals. Embryonic stem cells are pluripotent, allowing the differentiation of cell types from 3 germ layers. The establishment of a stable lineage of ESC is a valuable tool; however, some strains of mice are less permissive to ESC derivation or generation of chimeric animals (e.g. C57BL/6). Supplementation of culture medium with FCS, in the ESC derivation, may influence the potentiality to derivation or use of these strains in tetraploid complementation assays (Sato et al. 2009 Tsukuba Res. Inst. 47, 414–422). Thus, its replacement was carried out using knockout serum replacement (KSR®) to minimize the deleterious action of serum (Wang et al. 2007 Inst. of Biotech. 23, 269–272). Embryos were obtained from 5 females of lineage C57BL6/EGFP, aged between 21 and 30 days and weighing ∼35 g, and superstimulated according (Mancini et al. 2008 Transg. Res. 17, 1015). The animals were placed for mating with fertile males of the same strain in a proportion of one to one (male:female). The copulation was confirmed by plug vaginal (0.5 days postcopulation). Embryo recovery was performed 3.5 to 4.0 days postcopulation to obtain expanded (EB) or hatched blastocysts (HB). Zona pellucida was removed from EB with the aid of pronase solution, and the whole embryos (n = 8) were placed on a 4-well dish pretreated with pig skin gelatin 0.1%, under murine fibroblast primary in DMEM medium supplemented with 7.5% FCS and 7.5% KSR®, 10 mM βmercaptoetanol, 1 mM sodium pyruvate, 2 mM L-glutamine, and 83.4 mg mL–1 amikacin for 24 h. After this period, the medium was replaced by DMEM supplemented with 15% KSR®. The colonies began to grow between 3 and 6 days after in vitro culture of the embryos. Once established, the colony was picked and placed into new plates containing murine fibroblast primary every 48 to 72 h. After 14 days, the derivation was confirmed with some proved pluripotency markers by immunofluorescence (Oct3/4, SSEA-1, and Nanog) and karyotyping for ploidy detection. The reaction was positive for all tested markers in addition to the detection of the endogenous fluorescence from EGFP protein itself (C57BL/6EGFP origin). It was concluded that ESC derivation with partial serum replacement and using a less permissive strain such as C57BL/6EGFP is feasible, although with a reduced success rate (12.5%; i.e. 1 lineage – named BCM04 – from 8 attempts). Fellowships and grants were received from FAPESP, Brazil: 09/15919-4 (BCSC), 09/16254-6 (DMS), 09/17605-7 (CPG), 06/06491-2 (MFGN), and 07/07705-9 (MFGN).

148 EFFECTS OF Wnt3A SUPPLEMENTATION ON BOVINE BLASTOCYST CELL NUMBER AND ALLOCATION

Derivation of true bovine embryonic stem cells (ESC), as defined by their capacity to form robust teratomas and/or contribute to the germ line in chimeras, has not been achieved despite several attempts. It is possible that failures to derive bonafide bovine ESC are due to the inability of bovine embryonic cells to adapt to in vitro culture conditions that favor ESC derivation. Wnt pathways are involved in pluripotency and self-renewal of mouse and human ESC. Wnt signaling is also required for implantation competence in mouse blastocysts. Given the shared developmental potential between inner cell mass (ICM) and ESC, we hypothesized that Wnt could act on the ICM of bovine embryos increasing its proliferation potential. The objective of this study was to evaluate the effect of post-embryonic genome activation Wnt3A supplementation on blastocyst formation and cell allocation to ICM and trophectoderm (TE). In vitro fertilized bovine embryos at Day 4 of culture in KSOM medium were divided into 3 treatments: Control, no co-culture; co-culture with regular mouse embryonic fibroblasts (MEF); and co-culture with mouse L fibroblasts overexpressing Wnt3A protein (L-Wnt3A, Willert et al. 2003 Nature 423, 448-452). Embryos were cultured until Day 8 when blastocyst and hatching rates were recorded. Then, embryos were submitted to differential staining of ICM and TE by brief exposure to 0.25% Triton X-100 in PBS and staining with bisbenzimide and propidium iodide. Six IVF replications were performed and a total of 39 embryos were counted: 11 for Control, 16 for MEF, and 12 for L-Wnt3A. Only intact embryos after processing were used for cell count. Statistical analysis was performed by ANOVA using PROC MIXED of SAS software (SAS Institute Inc., Cary, NC, USA) in which each IVF was considered as a block with Tukey’s adjustment for mean comparison of rates and Bonferroni adjustments for mean comparison of cell counts. Results for blastocyst rate, hatching rate, ICM, TE, and total cell number are presented in the table below. Different superscript letters within columns indicate significant statistical difference (P < 0.05). These results indicate that L-Wnt3A fibroblast co-culture exerts a positive effect on bovine embryo cell number, resulting in a larger number of ICM cells in bovine embryos, which could be beneficial for ESC derivation attempts. Table 1.Blastocyst and hatching rates, ICM, TE, and total cell number results