Inducing pluripotency（4）
（http://www.stembook.org/node/514）
3. Cellular fusion
Fusion of various somatic cells and cell lines, generally using interspecies hybrids to distinguish genes expressed from each nucleus, has long been used to investigate phenotypic dominance at the cellular level. For instance, when fibroblasts are fused to myoblasts, are the resulting hybrids more like fibroblasts, myoblasts, or something in between？（Harris, 1965; Mevel-Ninio and Weiss, 1981; Wright, 1984）. Initial results indicating that some cellular identities could dominate over others in hybrids generated hope that this system could be used to investigate the mechanisms of this fate respecification as a proxy for understanding the effectors of cell fate decisions normally made in the process of development（Blau et al., 1985; Boshart et al., 1993）. However, between technical problems with interspecies fusion and the derivation of Embryonal carcinoma（EC; Martin and Evans, 1975）, and subsequently ES cells（Evans and Kaufman, 1981; Martin, 1981）, as more accurate in vitro models for cell fate determination, work with fusion waned considerably for several decades. Interest in this line of investigation was reinvigorated following the advent of mammalian NT and the subsequent speculation about the prospects of nuclear reprogramming for regenerative medicine. The observation that a pluripotent phenotype appeared to dominate following the fusion of murine somatic cells to EC（Miller and Ruddle, 1976）, Embryonic germ（EG; Tada et al., 1997）, and ES（Tada et al., 2003; Tada et al., 2001）cells seemed to promise that somatic-stem cell fusion might be an appealing alternative to inefficient and challenging NT. It was hoped that this system could be used for either the study of the mechanisms of nuclear reprogramming or perhaps eventually the direct production of patient-specific pluripotent stem cells. A report demonstrating that this capacity to reprogram somatic cells was conserved in human, as well as mouse, ES cells was further encouragement and represented the first demonstration of successful nuclear reprogramming of human somatic cells（Cowan et al., 2005）.
Despite this excitement, the fusion of ES and Somatic cells, as well as the subsequent Reprogramming, has proven to be quite inefficient（Cowan et al., 2005; Hochedlinger and Jaenisch, 2006; Tada et al., 2001）, limiting its usefulness in the study of the genetics and epigenetics of reprogramming. To date, therefore, investigators have focused on increasing these efficiencies by overexpressing genes already known to be important for pluripotency（Silva et al., 2006）, rather than providing new insights about pluripotency and reprogramming through fusion. Moreover, the problems associated with inefficiency are only compounded by the Tetraploid nature of the Hybrids generated by the Fusion itself. The presence of two complete genomes has severely limited the utility of this methodology for the study of reprogramming as well as presenting an enormous technical barrier to the production of autologous stem cells.
While the use of mature lymphocytes（which undergo a genetic rearrangement late in development）as the somatic fusion partner in the initial reports with mouse ES cells left no question that a terminally differentiated cell was being subjected to an ES cell environment（Tada et al., 2003; Tada et al., 2001）, determination of the extent of reprogramming has been less straightforward. Notably, the inability of tetraploid cells to contribute significantly to a chimeric embryo has limited the characterization of the pluripotency of the hybrid cells to less-stringent in vitro assays（Jaenisch and Young, 2008; Nagy et al., 1990; Tada et al., 2001）. Even more pressing, however, are questions regarding the state of the Somatic chromatin in the hybrid. Has the somatic nucleus truly been restored to a pluripotent state, or has it simply silenced the transcription of genes specific to the differentiated state, allowing the pluripotent ES nucleus control the hybrid cell's identity？Studies have sought to address this concern by demonstrating the Reactivation of the silent X-chromosome in hybrids formed with female somatic cells（Tada et al., 2001）, activation of reporter genes（Cowan et al., 2005; Tada et al., 2001）, the absence of appreciable DNA methylation at pluripotency-associated loci（indicating Demethylation occurred in the Somatic chromatin; Cowan et al., 2005）, and the expression of some Somatic-nucleus derived ES cell-associated genes by looking for specific Single nucleotide polymorphisms（SNPs）in hybrid cell transcripts（Cowan et al., 2005; Tada et al., 2003）. Although some studies have made use of relatively divergent strains of mice to facilitate these analyses（Tada et al., 2003）, genome-wide allele-specific expression analysis would help to elucidate the state of the somatic nucleus, but has yet to be performed with either mouse or human hybrid ES cells.
Tetraploidy also presents the most significant hindrance to the use of fusion in generating patient-specific stem cells as elimination of the ES cell genome after reprogramming will be necessary to produce autologous cells. The requirement for an ES cell nucleus was demonstrated directly by one study which used ultracentrifugation of ES cells（prior to fusion）to separate nuclear and cytoplasmic material. Pluripotent cell lines could be generated following Fusion of isolated nuclei（Nucleoplasts）with somatic cells, but Not following fusion to the enucleated cytoplasts（Do and Scholer, 2004）. While these authors concluded that ES cell chromatin was required for Reprogramming after hybrid formation, the successful NT results with metaphase-arrested zygotes discussed above（Egli et al., 2007）raises the possibility that mitotically arrested ES cell cytoplasts might also be capable of inducing a pluripotent state on somatic chromatin and may present a worthwhile avenue for future study. In addition to attempts to mechanically eliminate the ES cell chromatin with ultracentrifugation, a genetic system aimed at the same goal has also been developed, but to date only the elimination of a single chromosome has been demonstrated（Matsumura et al., 2007）. This system, depicted in Figure 4, makes use of Cre-mediated DNA recombination between sister chromatids to generate abnormal chromosomes which are eliminated during cell division. Although promising in principle, it remains unclear whether this technique could be used to simultaneously remove the entire ES-cell genome without introducing widespread genomic rearrangements and instability. Without an effective strategy for the disposal of the ES cell's genetic material, it may never be possible to use fusion to produce therapeutically relevant stem cells, nor for that matter, even to determine unambiguously whether the somatic chromatin has been fully reprogrammed.
Figure 4. Progress towards the elimination of ES nucleus following fusion, as described by Matsumura et al.（Matsumura et al., 2007）. A “Chromosome Elimination Cassette”（CEC）consisting of two oppositely-oriented LoxP sites flanking a GFP transgene is introduced into the ES cell genome in a single copy. Following DNA replication, the introduction of Cre mediates recombination between the CEC's on the two sister chromatids, yielding abnormal chromosomes with either no centromere（nullicentric）or two centromeres（dicentric）. During cell division, these abnormal chromosomes are naturally eliminated, thereby removing the ES cell-derived chromosome from the hybrid cell.
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