**2. Epigenetic reprogramming by SCNT**

such as DNA methylation, covalent histone modifications and chromatin remodeling. Once a cell's identity is set, it is difficult to convert it to other unrelated lineages, thus leading to stable and irreversible differentiated cell states. Nevertheless, there are a few exceptions of cell fate conversion during embryo development and adult tissue/organ regeneration, e.g. vascular endothelium to smooth muscle cells [2], which involve changing a cell's epigenetic signature

Apart from a few exceptions of natural cell fate conversion events, different strategies have been developed aiming to reprogram differentiated somatic cell fate to a pluripotent state (**Figure 1**). A historical strategy of reprogramming is by somatic cell nuclear transfer (SCNT) experiments. SCNT involves transplantation of a cell nucleus into an enucleated egg/oocyte in order to generate a "cloned" animal with an equivalent genetic composition as the donor individual. It is possible to derive embryonic stem (ES) cells from nuclear transplanted (NT) embryos (ntES cells), which shows indistinguishable pluripotent gene expression profiles

**Figure 1.** Strategies of reprogramming cell fate. Differentiated cells can be reprogrammed to pluripotent state by somatic cell nuclear transfer (SCNT), cell fusion, and ectopic expression of defined transcription factors. SCNT involves transplantation of a single differentiated cell nucleus into an enucleated egg/oocyte, which develops as a nuclear transplanted (NT) embryo. Cell fusion involves artificial fusion of a differentiated and a pluripotent cell to form a tetraploid pluripotent-like cell. Defined transcription factors (Oct4, Sox2, Klf4, c-Myc) can be ectopically expressed in

differentiated cells and convert them to induced pluripotent stem (iPS) cells.

into another unrelated kind.

166 Chromatin and Epigenetics

SCNT was first done by Briggs and Kings in 1952, who transplanted a blastula nucleus into an enucleated egg of the amphibian *Rana pipiens* [9]. Few years later, Gurdon *et al.* succeeded by using differentiated *Xenopus* intestinal epithelial donor nuclei for SCNT [10]. In 1997, the first cloned mammal, Dolly the sheep, was generated [11], and since then, more than 23 other mammalian species have now been successfully cloned [12]. Normal development of nuclear transplanted (NT) embryos requires recapitulation of the gene expression profile that supports the embryogenesis process by the differentiated donor nucleus. This involves re-activation of pluripotency genes, in particular *Oct4*, *Nanog* and *Sox2*, and repression of somatic lineage genes. In fact, the efficiency of reprogramming by SCNT is generally very low and less than 1% of NT embryos can develop into normal adults [13–15]. The cloned newborns often suffer from developmental abnormalities owing to incomplete reprogramming. It has been observed that *Oct4* was aberrantly expressed in cloned mouse blastocysts derived from cumulus donor nuclei [16, 17]. Besides, continuous expression of other somatic donor marker genes was demonstrated in some *Xenopus* NT embryos [18]. Some imprinted genes in donor cells were found to be aberrantly expressed in cloned embryos, presumably owing to the incomplete epigenetic reprogramming of the regulatory regions of imprinting loci [19–21]. Dysregulation of imprinted genes, such as *Igf2*, *Igf2r*, *H19*, and *Xist*, in cloned embryos can lead to both fetal and placental overgrowth and result in embryonic lethality or an abnormal growth condition called "large offspring syndrome", which is commonly found in cloned mammals [22–24]. Since the SCNT process does not increase the frequency of genetic alterations, it is suggested that the variable phenotypes observed in cloned embryos are associated with the reprogrammed epigenetic status of the donor nuclei [25]. This is supported by the findings that the developmental defects in cloned animals were not transmittable to the next offspring generation, indicating the presence of aberrant epigenetic reprogramming [26]. Aberrant DNA methylation patterns were indeed observed in NT embryos [27]. It was also demonstrated that the bovine NT blastocysts lack asymmetric patterns of both H3K9 methylation and acetylation between the inner cell mass and trophectoderm [28], which may account for abnormal cloned embryo development.

differentiated and condensed chromatin state of somatic nuclei with epigenetic modifying agents, e.g. 5-aza-deoxycytidine and trichostatin A (TSA), that facilitate chromatin relaxation [43–45]. Interestingly, the effect of TSA treatment in improving SCNT is associated with the reactivation of a subset of genes that are repressed by H3K9me3 in the somatic cells [46], presumably through introducing histone hyper-acetylation at their promoters. Altogether, SCNT provides a quick route of epigenetic reprogramming for a differentiated cell to a pluripotent state. Identification of the responsible reprogramming factors in the egg and oocyte cytoplasm will be one of the key future directions to improve the efficiency of SCNT and therapeutic

Resetting Cell Fate by Epigenetic Reprogramming http://dx.doi.org/10.5772/intechopen.81015 169

Cell fusion is a natural event that is crucial for fertilization and in various organs such as placenta, skeletal muscles and bones [47]. It has been proposed that the fusion of bone marrowderived stem cells and tissue cells, e.g. hepatocytes, is one of the mechanisms of tissue repair [48, 49]. Cell fusion experiments using pluripotent cells, e.g. an ES or embryonic germ (EG) cell, were shown to be able to reprogram a differentiated cell type [3–5]. Both ES and EG cells possess reprogramming ability and are able to reactivate pluripotent genes and silence differentiation genes in the somatic cell nucleus within a tetraploid hybrid cell after cell fusion. It is indeed the case that the new transcription profile of a hybrid cell is partly contributed by the reprogrammed somatic nucleus to a pluripotent-like state. Moreover, injection of hybrid cells into normal diploid blastocysts demonstrated their contribution to all three germ layers in the chimeras [4, 50], indicating the pluripotent nature of hybrid cells. Similar to the SCNT, different somatic cell types show different kinetics of reprogramming by the cell fusion approach,

which could be associated with the somatic chromatin accessibility status [51, 52].

polycomb repressive complexes in the cell fusion experiment using ES cells [57].

Cell fusion with a pluripotent cell can trigger extensive epigenetic reprogramming in the differentiated cell nucleus. It has been shown that reactivation of *Oct4* from the somatic nucleus occurs before DNA replication after cell fusion [53], suggesting the involvement of active demethylation process [54]. This was further supported by the functional roles of Tet1 and Tet2 in the demethylation of *Oct4* and imprinted control regions by fusing somatic cells with EG cells [55]. Besides, in a cell fusion experiment using thymocytes and ES cells from two different mouse strains, it was observed that the epigenetic profile of the somatic cell nucleus was reprogrammed to a similar pattern to that of the ES cell. Global histone H3 and H4 acetylation and H3K4 di- and tri-methylation were increased in the hybrid cell to a level comparable to ES cells, whereas these modifications are weak in the parental somatic thymocytes. Examination of gene specific loci showed that the *Oct4* promoter was enriched with H3 acetylation and the promoter of the thymocyte marker *Thy-1* was enriched with H3K27 tri-methylation in both ES cell and hybrid cell chromatin, whereas these epigenetic modifications are missing in the thymocyte [56]. Hence, the somatic genome has undergone epigenetic reprogramming triggered by fusion with the ES cell, suggesting that the process of cell fusion mediates a transcription activation-permissive chromatin state in the hybrid genome. In addition, silencing the somatic differentiation genes was shown to be associated with

cloning.

**3. Epigenetic reprogramming by cell fusion**

Although the rate of successful SCNT is very low, the reprogramming ability of factors in the egg/oocyte is highly efficient as the transplanted nuclei take less than 1 day to initiate cell division and trigger the "normal" developmental program. The donor cell epigenetic status has to be reprogrammed in order to support the embryonic program of development. In fact, genomewide demethylation was observed in the cloned blastocysts [29]. It has been shown that the *Oct4* promoter of somatic cells undergoes DNA demethylation after nuclear transplantation into the germinal vesicle of *Xenopus* oocytes [30]. This demethylation of the *Oct4* promoter was found to be mediated by Tet3 in a mouse SCNT study [31], which is essential for the reactivation of *Oct4* expression for successful SCNT. In addition, chromatin remodeling factors, such as ISWI and BRG1, are documented in facilitating the reprogramming of cell fate. It has been shown that ISWI, which is a chromatin remodeling ATPase, is able to dissociate TATA binding protein in somatic nuclei after incubation in the *Xenopus* egg extract, suggesting that ISWI-containing complexes are facilitating epigenetic reprogramming in an egg environment [32]. Besides, *Xenopus* egg extract depleted of BRG1 protein showed an abolishment of the reprogramming ability and hence failed to induce *Oct4* expression in the somatic nuclei [33]. Therefore, chromatin structure remodeling is believed to be one of the reprogramming mechanisms. In addition, the maternalderived histone H3 variant H3.3 in the enucleated egg was found to replace the canonical histone H3 in the donor nuclei after SCNT, leading to the reactivation of key pluripotent genes that are originally associated with repressive histone marks [34]. Histone H2A variant, macroH2A, also plays an important role in the reactivation of female donor cell's inactive X chromosome during reprogramming. It was shown that knockdown of macroH2A facilitates X-reactivation and the expression of pluripotent genes in cloned *Xenopus* embryos [35]. More recently, it has been demonstrated that the H3K9 tri-methylation (H3K9me3) of the donor cell genome is a major epigenetic barrier to SCNT. Ectopic expression of H3K9 demethylases *Kdm4b* or *Kdm4d* in the mouse donor cells de-repressed the genomic regions that are resistant to reprogramming and thus significantly improving SCNT efficiency [36, 37]. Similarly, removal of H3K9me3 by ectopic expression of other H3K9 demethylases also demonstrated improved reprogramming efficiency in human and bovine SCNT experiments [38, 39].

Interestingly, it has been shown that the epigenetic state of a differentiated cell is directly correlated with its reprogrammability [40]. SCNT with ES cell nuclei demonstrated a much higher efficiency of generation of NT blastocysts than using other somatic cell types [41]. This could be associated with a more relaxed chromatin configuration in ES cells that may render their epigenome more susceptible for reprogramming [42]. Alternatively, it has been demonstrated that the cloning efficiency can be significantly improved by pre-treating the more differentiated and condensed chromatin state of somatic nuclei with epigenetic modifying agents, e.g. 5-aza-deoxycytidine and trichostatin A (TSA), that facilitate chromatin relaxation [43–45]. Interestingly, the effect of TSA treatment in improving SCNT is associated with the reactivation of a subset of genes that are repressed by H3K9me3 in the somatic cells [46], presumably through introducing histone hyper-acetylation at their promoters. Altogether, SCNT provides a quick route of epigenetic reprogramming for a differentiated cell to a pluripotent state. Identification of the responsible reprogramming factors in the egg and oocyte cytoplasm will be one of the key future directions to improve the efficiency of SCNT and therapeutic cloning.
