**4. Epigenetic reprogramming by defined transcription factors**

In a groundbreaking discovery, Yamanaka *et al.* demonstrated that somatic cell state can be reprogrammed to a pluripotent state by the introduction of only four transcription factors; Oct4, Sox2, Klf4 and c-Myc, which are now also known as the Yamanaka factors. The first generation of iPS cells was obtained using a Fbx15-driven selection construct (Fbx15-iPS) and displayed a gene expression pattern very similar to that of normal pluripotent ES cells. However, the somatic epigenetic signature was only partially reprogrammed; the *Oct4* promoter, for example, retained some DNA methylation and no germline transmission was observed for these cells in chimeric mice. Hence these first-generation iPS cells were not fully pluripotent in nature [62]. Given the potential reprogramming capacity of these four factors, the second generation of iPS cells was generated by selection with a Nanog reporter construct (Nanog-iPS) [63, 64]. During the reprogramming process, the virally delivered transgenes were silenced, but on the contrary, the endogenous *Oct4* and *Sox2* loci were re-activated for the maintenance of pluripotency in iPS cells. In contrast to the Fbx15-iPS cells, these Nanog-iPS cells were able to undergo germline transmission in chimeric mice, and thus share this crucial feature of pluripotency with normal ES cells. These landmark studies that pioneered the derivation of mouse iPS cells led to the possibility of using the same strategy to generate human iPS cells. An initial study was performed by Thomas *et al.* in which a different combination of factors, OCT4, SOX2, NANOG and LIN28, was used to reprogram human fibroblasts into iPS cells [65]. Thereafter, Yamanaka and other groups succeeded in generating human iPS cells by using the same 4 Yamanaka factors as in the mouse iPS systems [66, 67]. To date, a number of different somatic cell types have been successfully reprogrammed into iPS cells, e.g. neural stem cells, keratinocytes, hepatocytes, gastric epithelia cells, pancreatic β cells, terminally differentiated B and T cells [8].

By using the defined transcription factor approach to reprogram cell fate, about 0.1–3% of the somatic starting cell population can be converted into iPS cells in around 2–3 weeks. The reprogramming efficiency is believed to be correlated with the differentiation state of the starting somatic cells. It has been shown that hematopoietic stem and progenitor cells can be reprogrammed to iPS cells 300 times more efficient than the terminally differentiated B and T cells [68]. Interestingly, partially de-differentiating mature B cells by either knockdown of *Pax5* or forced expression of *C/EBPα* resulted in efficient reprogramming by the Yamanaka factors [69], suggesting that the epigenetic status of differentiated cells is crucial in successful reprogramming. The dynamics of transcription profile and epigenetic patterns during the reprogramming process from somatic to iPS cells were studied in details. The reprogramming of somatic cell fate is a sequential stochastic event which involves a gradual silencing of the somatic lineage genes and the viral transgenes, and a sequential expression of alkaline phosphatase and SSEA1 in partially reprogrammed cells, whereas endogenous *Oct4* and *Nanog* are only activated in fully reprogrammed iPS cells. Induction of the four Yamanaka factors results in an immediate cellular response of inactivation of thousands of somatic lineage distal enhancers and, to a smaller extent, the H3K4me3-enriched somatic gene promoters [70, 71], leading to down-regulation of somatic identity genes. This initial phase of reprogramming is also accompanied by a global reduction of H3K27me3 resulting in loss of heterochromatin [72]. Meanwhile, mesenchymal transcription factors, such as *Snail1/2*, *Zeb1/2*, are repressed [70, 73, 74], whereas epithelial transcription factors, such as *Cdh1*, *Epcam*, are activated [75, 76], resulting in mesenchymal-epithelial transition (MET). This is associated with an increase in H3K4me2 at epithelial genes, but a decrease in H3K4me2 and H3K79me2 at mesenchymal genes [71, 77]. Despite an increase in global H3K36me2/3 level, loss of H3K36me2/3 was observed at the *Ink4-Arf* locus, leading to enhanced cell proliferation during reprogramming [78]. The following phase of reprogramming is marked by upregulation of endogenous pluripotency genes to establish transcriptional program, which is independent of transgene expression [73, 79]. The final phase of reprogramming involves elongation of telomeres, X-reactivation in female iPS cells, and upregulation of DNA methylation genes [79]. This coincides with loss of DNA methylation and downregulation of *Xist* expression in the somatic inactive X chromosome [80, 81].

Reprogramming to pluripotency by cell fusion approach requires lengthy selection of the successfully reprogrammed hybrid cells. The reprogramming efficiency of cell fusion is usually less than 0.001%, depending on the somatic cell types [50, 58]. The low reprogramming efficiency in hybrid cells can be largely enhanced by manipulation of key pluripotency-associated genes like *Nanog* [58, 59] and *Sall4* [60], or by activation of the Wnt signaling pathway [61], emphasizing the importance of these factors in cell fusion reprogramming. Overexpression of *Nanog* or *Sall4* in ES cells demonstrated a several hundred-fold increase in reprogramming efficiency after cell fusion. Similarly, treatment of ES cells with Wnt3a for 24–48 hours enhanced the reprogramming of somatic cells by 20-fold. However, owing to the low reprogramming efficiency and the tetraploid genome of the resulting hybrid cells, reprogramming

by the cell fusion approach becomes less promising in regenerative medicine.

**4. Epigenetic reprogramming by defined transcription factors**

ferentiated B and T cells [8].

170 Chromatin and Epigenetics

In a groundbreaking discovery, Yamanaka *et al.* demonstrated that somatic cell state can be reprogrammed to a pluripotent state by the introduction of only four transcription factors; Oct4, Sox2, Klf4 and c-Myc, which are now also known as the Yamanaka factors. The first generation of iPS cells was obtained using a Fbx15-driven selection construct (Fbx15-iPS) and displayed a gene expression pattern very similar to that of normal pluripotent ES cells. However, the somatic epigenetic signature was only partially reprogrammed; the *Oct4* promoter, for example, retained some DNA methylation and no germline transmission was observed for these cells in chimeric mice. Hence these first-generation iPS cells were not fully pluripotent in nature [62]. Given the potential reprogramming capacity of these four factors, the second generation of iPS cells was generated by selection with a Nanog reporter construct (Nanog-iPS) [63, 64]. During the reprogramming process, the virally delivered transgenes were silenced, but on the contrary, the endogenous *Oct4* and *Sox2* loci were re-activated for the maintenance of pluripotency in iPS cells. In contrast to the Fbx15-iPS cells, these Nanog-iPS cells were able to undergo germline transmission in chimeric mice, and thus share this crucial feature of pluripotency with normal ES cells. These landmark studies that pioneered the derivation of mouse iPS cells led to the possibility of using the same strategy to generate human iPS cells. An initial study was performed by Thomas *et al.* in which a different combination of factors, OCT4, SOX2, NANOG and LIN28, was used to reprogram human fibroblasts into iPS cells [65]. Thereafter, Yamanaka and other groups succeeded in generating human iPS cells by using the same 4 Yamanaka factors as in the mouse iPS systems [66, 67]. To date, a number of different somatic cell types have been successfully reprogrammed into iPS cells, e.g. neural stem cells, keratinocytes, hepatocytes, gastric epithelia cells, pancreatic β cells, terminally dif-

By using the defined transcription factor approach to reprogram cell fate, about 0.1–3% of the somatic starting cell population can be converted into iPS cells in around 2–3 weeks. The reprogramming efficiency is believed to be correlated with the differentiation state of the starting somatic cells. It has been shown that hematopoietic stem and progenitor cells can be reprogrammed to iPS cells 300 times more efficient than the terminally differentiated B and T cells [68]. Interestingly, partially de-differentiating mature B cells by either knockdown of A number of epigenetic remodeling factors are involved in the reprogramming events. Both polycomb (PcG) and trithorax (TrxG) group proteins were found to be crucial in the derivation of iPS cell colonies. Upon knockdown of *Wdr5*, which is a core component of TrxG protein complex, cells failed to establish H3K4me3 at the pluripotent genes, like *Oct4* and *Nanog*, for their reactivation [82]; whereas inhibition of the core components of the polycomb repressive complex 1 and 2 reduced reprogramming efficiency [83], partly because of the dysregulation of genes involved in the MET process [84]. This is similar to the findings that inhibition of H3K79 methyltransferase DOT1L facilitates the loss of H3K79me2 mark at the mesenchymal genes to promote MET during reprogramming [83]. The H3K27 demethylase Kdm6a (also known as Utx) can directly interact with Oct4/Sox2/Klf4 to remove the repressive H3K27me3 mark from the early pluripotent genes in somatic cells for their reactivation [85]. This is in agreement with the findings that depletion of histone H2A variant, macroH2A, enhances reprogramming, owing to its co-occupancy with H3K27me3 to repress pluripotent genes [86, 87]. Besides, the H3K36 demethylase Kdm2b (also known as Jhdm1b) enhances the activation of early responsive genes (*Cdh1*, *Epcam*, *Dsg2*, *Dsp*, *Irf6*) during reprogramming through the removal of H3K36me2 at their promoters [88]. Interestingly, H3K9me3 was also found to be one of the major epigenetic barriers in the generation of iPS cells [89], similar to the findings in SCNT experiments. Depletion of the H3K9 methyltransferases SUV39H1/H2, Ehmt1/2 and Setdb1 or inhibition of Cbx3, a protein that recognizes H3K9 methylation, enhances reprogramming by de-repressing *Nanog* and abolishing the cellular responses to BMP signaling [89–91]. Although Dnmt3a/b were found to be dispensable [92], DNA demethylation of key pluripotency loci mediated by Tet proteins is required for efficient reprogramming [93–95].

inactive X chromosome of the donor cell is preferentially chosen for inactivation in the trophectoderm, which is in contrast to the random X inactivation in the embryo proper, demonstrating a certain extent of memory of the inactive X chromosome status [48]. Random X inactivation in the placenta was found in deceased cloned bovine embryos, which suggests that the persistence of this inactive X chromosome memory in the placenta may be crucial for fetal survival [49].

Resetting Cell Fate by Epigenetic Reprogramming http://dx.doi.org/10.5772/intechopen.81015 173

The epigenetic memory in NT embryos that maintains the donor expression gene states can be explained by the inheritance of DNA methylation patterns. Heritability of DNA methylation is mediated by the activity of the maintenance DNA methyltransferase Dnmt1 which preferentially targets hemi-methylated DNA. Thus, Dnmt1 restores the parental methylation status on the newly synthesized daughter DNA strand, thereby maintaining a silent gene state after cell division [108]. Methylated donor genes, such as pluripotency genes, remain inactivated after SCNT, apparently owing to the persistent donor-specific methylation pattern in the cloned embryos, possibility mediated by the residual somatic form of Dnmt in the donor nucleus. DNA methylation therefore provides a plausible mechanism for the propagation of a silent memory state in SCNT [109]. On the other hand, an active gene memory of the donor differentiation state is also observed in NT embryos. For example, both the donor endoderm and neurectoderm markers, *Edd* and *Ncam*, were found to be aberrantly expressed in *Xenopus* NT embryos derived from the respective donor cell types [18]. This active gene memory was further demonstrated to be associated with the incorporation of a histone H3 variant H3.3 at the active gene loci in *Xenopus* NT embryos [110]. Histone variant H3.3 is enriched in the regulatory region of active genes and is preferentially marked by modifications associated with an active chromatin state, such as H3K4 methylation, H3K9 acetylation and H3K79 methylation [111, 112]. Experiments using a mutant form of H3.3 demonstrated that the K4 residue on H3.3 plays a key role in the inheritance of active epigenetic memory [110], proposing a model in which H3.3 K4 methylation creates an "active histone environment" for the recovery of

active chromatin configuration in daughter cells after chromosomal replication [113].

Early studies of iPS cells demonstrated that the Nanog-iPS cells displayed not only a highly similar transcriptome to wild-type ES cells, but also an ES cell histone modification profile. Genome-wide comparison of histone modifications (H3K4 and H3K27 trimethylation) between ES cells, MEFs and MEF-derived iPS cells demonstrated that more than 94% of the ES- or MEF-signature genes in iPS cells have identical histone methylation marks as in ES cells. Only 0.7% of these signature genes retain the histone methylation status of the original MEFs [63]. However, other gene expression profile studies of iPS cells showed that a significant number of differentiation genes have a similar expression pattern to that in the somatic cell of origin, but not in ES cells [114–117]. This transcriptional memory in iPS cells was found to be correlated with biased differentiation towards the original cell lineage, and with less competence in differentiation to other unrelated somatic lineages [115, 118–121]. Importantly, the persistent expression of somatic genes in iPS cells was associated with the somatic DNA methylation pattern [117, 122–124], highlighting the crucial role of epigenetic regulation in the retention of memory. This is in fact similar to the observation that an incomplete removal of donor cell DNA methylation pattern was observed in some aberrantly developed NT embryos [125]. Strikingly, the addition of epigenetic modifying agents, such as DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), can enhance the iPS cell reprogramming efficiency [70]

Previous studies demonstrated that the partially reprogrammed iPS cells contained significantly fewer genes marked by the bivalent chromatin signature (co-existence of both H3K4 and H3K27 methylation) and an enrichment of DNA hyper-methylated loci when compared to the wild-type ES cells and the fully reprogrammed iPS cells [70]. Therefore, it is proposed that completion of the epigenetic reprogramming process is pre-requisite for the acquisition of pluripotency. This is supported by the observation that treatment of partially reprogrammed iPS cells with the DNA methyltransferase inhibitor 5-aza-cytidine was able to promote their transition into the fully reprogrammed pluripotent state [70]. Besides, inhibition of H3K27 methyltransferase Ezh2 by small molecule, GSK-126, reduced reprogramming efficiency [84], whereas inhibition of DOT1L by small molecule, EPZ004777, showed enhancement of reprogramming [83]. Various histone deacetylase inhibitors (HDACi) were also shown to improve reprogramming [96–100]. In combination with HDACi valproic acid, human iPS cells can be generated only with Oct4 and Sox2 with a comparable reprogramming efficiency by the four Yamanaka factors [101]. Interestingly, it was found that vitamin C can increase reprogramming efficiency by promoting the transition of pre-iPS cells to fully reprogrammed cells [102], potentially through acting as a cofactor of Kdm2b to induce H3K36me2/3 demethylation [78], activation of H3K9 demethylases (Kdm3a, 3b, 4c and 4d) to remove H3K9me3 [89], and promoting Tet-mediated DNA demethylation [103]. With the aid of small chemical molecules, the iPS cell reprogramming efficiency and duration could be further improved.
