muscarinic receptor antagonists br Materials and methods br

Materials and methods
Results
Discussion The study of human embryonic development is challenged by ethical concerns associated with the use of in vitro fertilized donor material, or abortions, and by the limited access to such sample material. Consequently, the ultrastructural information about human epiblasts at the post-implantation stage is primarily based on findings from other species such as other primates, pigs and rodents (reviewed in (Pera and Trounson, 2004)). Here, we analyzed porcine gastrulation by TEM in order to decipher whether the MET occurring on the road to induced pluripotency mimics a reversal of the EMT which takes place during gastrulation in vivo. We were able to demonstrate that the artificial rewinding of differentiated human muscarinic receptor antagonists to induced pluripotency results in a cellular phenotype which resembles the human epiblast 7–12days post-fertilization (Gasser, 1975; Hertig and Rock, 1945, 1949a, 1949b, 1949b). In this regard, one may speculate that the apical-to-basal migration of gastrulating epiblast cells during EMT is recapitulated in reverse order in iPSC colonies around day 12, when sporadically distributed mesenchymal-like cells underlying the early forming iPSC epithelium appear to move in the basal–apical direction up through the discontinuous basement membrane to become integrated as epithelial cells into the forming colony (Fig. 2, D12). The hypothesis that fibroblasts dedifferentiate to iPSC in a process which bears resemblance to a reverse gastrulation was recently supported by a publication from the Yamanaka group (Takahashi et al., 2014). They showed that TRA-1-60 positive human cells undergoing reprogramming to iPSC had a transient gene expression profile reminiscent of primitive streak mesendoderm, and that overexpression of the transcription factor forkhead box H1 (FOXH1), which is crucial for the specification of the primitive streak, enhanced MET and the formation of iPSC (Takahashi et al., 2014). It would be interesting to visualize the morphogenesis of reprogramming cells which display this transient mesendodermal gene expression, in order to establish whether they resemble mesendodermal progenitors in the primitive streak on the morphological level. In present study, we were primarily analyzing cells in earlier stages of reprogramming between D0 and D12, when no pluripotency markers were yet expressed, hence we cannot exclude the possibility that our ultrastructural characterizations were performed on partially reprogrammed cells. The correlation between acquisition of epithelial and pluripotent characteristics shown in Table 1 suggests that the epithelial iPSC-like colonies presented in Fig. 2 (D12, D18) were indeed destined to become true iPSCs. However, in order to fully confirm this, correlative light and electron microscopical analyses of TRA-1-60 positive cells undergoing reprogramming from D12 and throughout the maturation period would be needed. Gene expression studies of partially or fully reprogrammed MEFs have led several groups to define distinct stages of reprogramming, as elegantly reviewed by David and Polo (2014). Generally, three phases are proposed; the first one being defined by MET, increased proliferation and cytoskeletal rearrangements (Li et al., 2010; Samavarchi-Tehrani et al., 2010; Sakurai et al., 2014; Mikkelsen et al., 2008; Sridharan et al., 2009; Stadtfeld et al., 2008; Polo et al., 2012; Hansson et al., 2012), the second comprising an intermediate or partial stage of reprogramming during which early pluripotency markers are upregulated (SSEA1 in mouse, TRA-1-60 in human, followed by Nanog and Oct4) (Sridharan et al., 2009; Stadtfeld et al., 2008; Polo et al., 2012; Hansson et al., 2012; Brambrink et al., 2008; Tanabe et al., 2013), and finally the third phase when passaged pluripotent colonies exhibit transgene independence, X-reactivation and telomere elongation (Mikkelsen et al., 2008; Sridharan et al., 2009; Stadtfeld et al., 2008; Brambrink et al., 2008). As previously mentioned, these three phases were named Initiation, Maturation and Stabilization by J. Wrana and group in 2010 (Samavarchi-Tehrani et al., 2010.