We hypothesized that specific epigenetic

We hypothesized that specific epigenetic determinants on the level of DNA methylation and histone modifications might underlie the remarkable differentiation potential of USSC. Indeed, the present work reveals an unexpected epigenetic heterogeneity with respect to the DNA methylation patterns covering the OCT4 –5′ region and the NANOG promoter. In particular, we observed subpopulations of USSC monensin Supplier with partially methylated, weakly methylated, and sporadically unmethylated OCT4 and NANOG promoters. In line with that, residual weak OCT4 transcription is detectable, presumably arising from the few unmethylated cells. This epigenetic variability could thus reflect a certain variability in differentiation capacity among USSC lines as described recently (Liedtke et al., 2010). In particular, less methylated USSC cells might reside in a very early differentiation state and may therefore possess extended self-renewal and broad differentiation potential. However after 14days of osteogenic differentiation a minor increase of DNA methylation was observed at the OCT4 5′ region, underlining a direct correlation between DNA methylation and differentiation state (data not shown). Along the same lines, the hTERT promoter was found to be unmethylated in USSC. However, the DNA methylation state of the extended CpG island overlapping the promoter region showed a more complex pattern combining characteristics of ES cells with those of differentiated fibroblasts. Again, acknowledging that hTERT is not expressed in USSC (Aktas et al., 2010), the hTERT gene nonetheless is in an epigenetically uncommitted state that mirrors the uncommitted differentiation state of the USSC. Strikingly, the SOX2 promoter and main enhancer were completely unmethylated, although no trancripts were detectable. Since in ES cells the same methylation status correlates with strong expression it can be hypothesized that either an appropriate activator is missing or a repressor is present in USSC. In addition, in some USSC lines the SOX2 promoter exhibited a bivalent histone modification signature, combining the opposing active and repressive histone marks dimeH3K4 and trimeH3K27. A bivalent histone signature is typically found in pluripotent stem cells and is indicative of genes that can be either activated or silenced, depending on the differentiation pathway the cell starts to pursue (Gan et al., 2007). Notably, on overexpression of OCT4, a known positive regulator of SOX2, SOX2 transcription was inducible within 48h. Thereby at the SOX2 promoter the ratio between the repressive trimeH3K27 and the activating dimeH3K4 histone modification turned in favor of the latter (data not shown). This demonstrates that the “poised” state of the gene can be resolved toward an active state using an appropriate stimulus. It cannot be excluded that the observed bivalent histone signatures are based on a mixture of USSC with opposing histone modifications in the same cell preparation. On the other hand, the clonal origin of the cells and the stable phenotype across many cell divisions makes this possibility rather unlikely. It appears to be more likely that our data reflect true bivalent signatures in the respective USSC lines, as had been originally described by Lander and colleagues for ESC (Bernstein et al., 2006). The bivalent epigenetic signature of SOX2 could be a crucial prerequisite for the ability of the USSC to enter the neural differentiation pathway (Kögler et al., 2004). An appropriate activator acquired in a later differentiation stage could induce SOX2 expression and resolve this uncommitted bivalency. For proper neurogenesis Sox2 expression is of fundamental importance (Ferri et al., 2004). Thereby strong gene expression in neural stem/progenitor cells is driven by the Sox2 regulatory region 2 (SSR2) which in turn is bound by class III POU proteins, such as Brn1 and Brn2 and also Sox2 itself (Miyagi et al., 2006). It is now well established that somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by ectopic overexpression of stem cell factors like OCT4, SOX2, and NANOG (Zaehres et al., 2010a,b), and it is also known that epigenetic reprogramming is a rate-limiting step for successful generation of iPS (Takahashi and Yamanaka, 2006; Mikkelsen et al., 2008). The uncommitted epigenetic signatures of OCT4, SOX2, and NANOG as presented here suggest that USSC might be endowed with a certain degree of epigenetic plasticity and thus may be a valuable source for the generation of iPS cells from cord blood. The young age of cord blood cells, the lack of virus infections, and the fact that neonatal cells are available from large cord blood banks make USSC a clinically highly desirable source for iPS generation. Indeed, it was recently shown that four-factor reprogramming of USSC to iPS is possible (Zaehres et al., 2010a,b). It could thus be speculated that the incomplete DNA methylation and reversible histone modification signatures of key pluripotency genes in these cells would facilitate the reprogramming process. Moreover, the fact that overexpression of OCT4 leads to rapid reactivation of SOX2 (Fig. 3) suggests that the reprogramming of USSC might be possible without overexpression of SOX2, which is currently under investigation. In general, the knowledge of the factors defining these properties will provide guidance for novel direct differentiation protocols ex vivo and hopefully novel therapeutic applications in regenerative medicine.