The lack of correlation between

The lack of correlation between ESC derivation efficiency and both the moment when the cleavage at two-cell stage occurs and the cleavage plane orientation could be related to the isolation of blastomeres prior to the derivation of the lines. It is well known that the developmental program is so flexible that blastomere fate can be changed when subjected to experimental manipulation such as isolation from the embryo or blastomere reaggregation, situations that disturb blastomere environment (niche) inside the embryo. Individual potential of blastomeres inside the embryo depends on morphological, molecular and epigenetic changes which play a central role at specific moments of the preimplantational development (Yamanaka et al., 2006; Marikawa and Alarcon, 2009). Once a single blastomere is removed from the embryo, morphological aspects such as position of blastomeres and cell–cell contacts are disturbed, so it is possible that the isolated blastomere has enough plasticity to readdress its developmental program by entering a transient moment of a higher developmental potential than within the embryo that could favor ESC derivation. But blastomere fate, as previously mentioned, is also guided by molecular aspects such as transcription factors and epigenetic marks. If these molecular footprints remained stable in the isolated blastomere, i.e., a predominant expression of characteristic ICM or TE transcription factors or non-modified epigenetic marks, they would continue guiding the isolated blastomeres to their initial fate inside the embryo. We wanted to further analyze differences between ESC lines derived from these groups of blastomeres by the in vitro differentiation experiments. We did not detect differences regarding to the expression of differentiation markers since all ESC lines analyzed showed expression of early differentiation markers to melanotropin of the three germ layers. Further analysis such as chimeric contribution and germline transmission of the ESC lines will be of interest to address possible differences between ESC lines derived from these groups of blastomeres. Immunostaining of pseudoblastocysts, based on the expression of a stem cell marker, was performed to determine whether each isolated blastomere was equally able to form a visible ICM and, thus, produce an ESC line. Data obtained from this experiment correlate with the aforementioned results about ESC derivation since 2/3E, 2/3M and 2/4 blastomeres showed equal competence to form ICM cells, the cell lineage that will give rise to ESC lines. Moreover, pseudoblastocysts formed contained similar mean numbers of ICM and TE cells, further confirming the plasticity and the acquisition of a higher developmental potential once blastomeres are isolated from the embryo based on the premises discussed above. These results are consistent since all of these groups developed into a pseudoblastocyst starting from the same number of isolated blastomeres and they are derived from embryos at equivalent developmental stages, three- and four-cell stages which originate from an asynchrony of cleavage divisions at the two-cell stage to give rise to four-cell stage embryos. Our data are in concordance with the study reported by Piotrowska-Nitsche et al.(2005) in which they propose that all four-cell blastomeres can have full developmental potential although they differ in their individual developmental properties within the embryo. Despite this, our results do not agree with those reported by Lorthongpanich et al. (2008) as they observed that not all single blastomeres from two- and four-cell stages of outbred CD-1 mouse embryos were able to form ICM cells. This discordance between both studies could be due to the use of a different mouse strain since it is well known that the inbred mouse strain 129/Sv provides the highest efficiencies of ESC derivation (Tesar, 2005; Wakayama et al., 2007), thus being the most commonly used strain to derive mouse ESC lines. Moreover, unlike in our study, Lorthongpanich et al. (2008) used SOX2 as an ICM and stem cell marker. Despite SOX2 is expressed in a similar manner as OCT4 in these cells, OCT4 expression is thought to be higher in ICM cells (Koestenbauer et al., 2006). Differences in expression and detection thresholds of both pluripotency makers in the ICM of pseudoblastocysts may also explain differences found between both studies.