br Materials and methods br Acknowledgment br Introduction C

Materials and methods
Acknowledgment
Introduction Cellular differentiation and dedifferentiation require global changes in gene expression regulated in part by epigenetic mechanisms such as DNA methylation and chromatin reorganization. DNA methylation is a covalent modification of cytosine at position C5 in CpG dinucleotides. By computational prediction it has been estimated that 29 000 CpG-rich regions are distributed in the human genome, many of them spanning the promoter and first exon of approximately 60% of our genes (Bird, 2002). Typically, if they are methylated the corresponding gene promoter is constitutively inactivated (Paz et al., 2003). The methylated state, once established, is stable for many cell divisions (Bird, 2002). Another crucial epigenetic determinant of proper gene regulation in developmental processes is a dynamic chromatin structure. Active genes are associated with an acetyl group on lysine 9 of histone 3 (acH3K9), on lysine 8 of histone H4 (acH4K8), and methyl groups on lysine 4 of histone H3 (meH3K4) (Jenuwein and Allis, 2001; Kouzarides, 2007). On the other hand, inactive genes bear either a mark for constitutive heterochromatin such as dimethylation on lysine 9 of histone H3 (dimeH3K9) (Bannister et al., 2001; Nakayama et al., 2001; Noma et al., 2001) or a mark for facultative heterochromatin, which is trimethylation on lysine 27 of histone H3 (trimeH3K27) (Plath et al., 2003). Genome-wide epigenetic marks must be heritable and dynamic, in particular during early development where they ensure differentiation state-specific gene regulation (Amabile and Meissner, 2009). The master transcription regulators NANOG, OCT4, and SOX2 form a basic regulatory network that governs pluripotency and self-renewal in human embryonic stem SAR 405 (ESC) (Boyer et al., 2005). NANOG deficiency in ESC leads to loss of self-renewal capability (Chambers et al., 2003; Mitsui et al., 2003) and predisposes the cells to uncommitted differentiation (Chambers et al., 2007). A critical amount of OCT4 is required to sustain self-renewal of ESC, whereas up- or downregulation induces divergent developmental programs (Niwa et al., 2000). SOX2 maintains pluripotency in ESC and in neural stem cells (Yuan et al., 1995; Masui et al., 2007). It is well established that the genes encoding the three key regulators of stemness all bear differentiation state-specific epigenetic marks, which undergo changes during differentiation. Another hallmark of pluripotent stem cells is telomerase activity. In ESC, active telomerase is able to maintain telomere length whereas in proliferative somatic stem cells a tightly regulated telomerase activity seems not to be sufficient to fully maintain it (Shay and Wright, 2010). Pluripotent unrestricted somatic stem cells (USSC) from human umbilical cord blood reside in an early differentiation state, can be propagated to high cell numbers, and on treatment with appropriate stimuli display broad differentiation capabilities in vitro and in vivo (Kögler et al., 2004, 2006). They thus represent promising candidates for regenerative and cell replacement therapies. However, an indispensable prerequisite to employ USSC for such purposes in a controlled manner is a better understanding of the molecular mechanisms that are necessary for establishment and maintenance of their stem cell properties. The major stem cell factors OCT4, NANOG, SOX2, (Kluth et al., 2010), and hTERT (Aktas et al., 2010) are all not detectable in USSC at the protein level. Nevertheless, we hypothesized that the stem cell characteristics of USSC could be mirrored by a specific epigenetic signature of these major regulators of pluripotency.
Results
Discussion Cord blood contains hematopoietic, mesenchymal, and embryonic tissue-like stem cells with considerable proliferative potential, self-renewal abilities, and various in vitro differentiation capabilities on stimulation with appropriate cues (Harris, 2009). It is further hypothesized that cord blood stem cells might participate in fetal organogenesis (Ratajczak et al., 2008). In fact, somatic stem cells from human umbilical cord blood display broad differentiation capacity in vitro (Kögler et al., 2004).