Reproduction in Domestic Animals

Farm Animals Embryonic Stem Cells 1952006). In practical terms, this translates in no definedepiblast being present in these species before hatchingwhich in vivo occurs on late day 6 or on day 7 ofdevelopment in pig and days 8–9 in cow (Hunter 1974;Renard and Heyman 1979; Betteridge and Flechon1988). At this stage the ICM is present, defined as thecells comprised between the Rauber’s layer, separating itfrom the uterine lumen, and the visceral endoderm.Which is to say that, in order to obtain in vivo producedpig and cow blastocysts equivalent to their murinecounterparts, the uterus must be flushed on days 7–8and 8–9, respectively, when embryos are completelyhatched and expanded (Betteridge and Flechon 1988;Vejlsted et al. 2006). Upon hatching, pig blastocystsretain a round shape and during days 8 and 9, thehypoblast is formed from the ICM and graduallyproliferates as a confluent cell layer surrounding theblastocoel cavity. At the same time and through day 10in pig and day 12 in cow, the polar trophectoderm thatcovers the epiblast, referred to as the Rauber’s layer,begins to degenerate until is completely lost leaving theepiblast directly exposed to the uterine lumen (Maddox-Hyttel et al. 2003; Flechon et al. 2004). The area wherethe epiblast has surfaced is spherical, has a whitishcolour under the stereomicroscope and is referred to asthe embryonic disc whose internal surface is covered bycuboidal hypoblast cells. Between days 11 and 13,development continues with the embryonic disk and thewhole conceptus begins to assume an ovoid shape. Atthis stage, the first signs of polarity become visible alsoat the stereo microscope in the form of a crescentshapedthickening on the posterior third (Vejlsted et al.2006). This thickening will originate, within a couple ofdays, the primitive streak which accompanies theappearance of defined mesoderm and endoderm layers.This differs from the mouse where mesoderm andendoderm differentiation follows rather than precedesthe primitive streak formation (Tam and Behringer1997). Around days 13 and 14 while the primitive streakis still clearly visible, the major part of the epiblasttransforms into neural ectoderm and forms the neuralplate. This corresponds to a gradual down regulation oftypical pluripotency maker OCT4 which is substitutedby b-tubulin III expression, a marker of neural differentiation.Therefore, it can be assumed that embryos atthis stage are no longer suitable for ESC derivation. Insummary, the extended pre-implantation period togetherwith the formation of an embryonic disk makesungulate embryo epiblast available for a much longertime than in rodents and primates. As a consequence,the ICM available in the pre-hatching blastocyst maynot be exactly equivalent to the mouse epiblast usuallyrecovered for ESC derivation.However, it must be noted that, in pig and cow, a bigvariation in size is observed in embryos collected at thesame time and embryos of the same size showsubstantial differences in the development of theembryo proper. This means that, when post-hatchingembryos are used for ESC derivation, the simpleindication of the day of collection may comprise awide range of development. This wide variation mayexplain why a recent survey of the literature hasshowed no obvious effect of embryo age on theefficiency of ESC line derivation, at least in pig (Breviniet al. 2007b).Although derivation efficiency was not affected by theage of the embryo, the quality of the cell lines mighthave been different as suggested by some recent findingsin mouse. Two independent groups, in fact, showed thatmESC derived from early post-implantation embryoscorrespond more closely to hESC rather than mESC, inall aspects tested so far (Brons et al. 2007; Tesar et al.2007) including being leukemia inhibitory factor (LIF)and bone morphogenetic protein-4 (BMP4) independentbut requiring activin and FGF for efficient self-renewal.Interestingly, these cell lines, named epiblast stem cells(EpiSCs) were unable to form chimeras (Tesar et al.2007) or did so at a very low efficiency (Brons et al.2007) closely resembling, in this respect, to domesticanimal cell lines. Moreover, EpiSCs did not showdependency from a specific genetic background similarlyto human and farm animal cell lines.Therefore, differences in pre-attachment developmentand timing of isolation may be another factor added togenetic variation explaining species-specific differencesin ESC derivation efficiency and pluripotency.It has been speculated that cells able to give rise to‘true’ ESCs are overgrown by more rapidly dividingslightly later cells (Lovell-Badge 2007) and this wouldexplain the differences between those few mouse strainswere ESC derivation is possible and all other speciesincluding human.How Different Are ESCs from DifferentSpecies?Recent evidence, based on the comparison between thetrascriptomes of mESC and hESC, leads to the conclusionthat mESCs are substantially different from humancell lines. In fact, combining together the results ofdifferent studies, it appears that only 13–55% oftranscripts enriched in mESCs are also enriched inhuman lines but when comparison is performed amongdifferent hESC lines the overlap raise to 85–99% (seeEckfeldt et al. 2005; for detailed references). Thesubstantial differences determined between hESC andmESC expression profiles are followed by some morphologicaland functional differences between the celllines of the two species (Laslett et al. 2003; Ginis et al.2004; Rao and Zandstra 2005). For instance, hESCstypically grow in tightly adherent, flattened groupsrather than in rounded clumps and tolerate physicalseparation very poorly. ESCs of both typically grow wellin presence of mouse embryonic fibroblasts; hESCs donot require the presence of LIF to activate JAK-STAT3transcription factors in order to maintain their pluripotencyin culture. ESCs of both the species can bemaintained in culture without a feeder layer, but in thiscondition hESCs require the presence of fibroblastgrowth factor-2 and activin, whereas mESCs need LIFand bone morphogenetic protein-4 (BMP4). In contrast,BMP4 induces trophoblast differentiation in hESCs(Ludwig et al. 2006; Brons et al. 2007; Tesar et al.2007). Furthermore, although they express the same setof factors known to be required for pluripotency inmouse ESCs (such as OCT4, SOX2 and NANOG),Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag