**4. Induced pluripotent stem cells**

Takahashi and Yamanaka [37] discovered that the genome of a differentiated somatic cell can be epigenetically reprogrammed to pluripotency by the induced expression of pluripotency transcription factors, resulting in the generation of pluripotent stem cells [induced Pluripotent Stem Cells (iPSCs)]. These authors initially expressed 24 pluripotency genes in fetal fibroblasts to reprogram them into pluripotent cells, and subsequently found that the expression of only 4 of these genes, namely *Oct4, Sox2*, *c-myc*, and *Klf4*, was sufficient to achieve the same results. Nanog was dispensable for the cellular reprogramming. In addition, a year later, Okita et al. [38] produced germline-competent mouse iPSCs using the same growth factors, but by selecting the reprogrammed cells by Nanog expression.

The production of human iPSCs started shortly after the publication of the first report of mouse iPSC derivation. Thus, it was not surprising that the establishment of the first human iPSCs was reported by two groups simultaneously. The laboratory that produced the first mouse iPSCs successfully generated iPSCs from adult human fibroblasts by using the same 4 transcription factors employed in the initial experiment [39]. The other group, from the laboratory of human ESC pioneer James Thompson, was able to reprogram human fibroblasts by expressing OCT4, SOX2, NANOG, and LIN28 [40].

Predictably, these studies sparked significant interest among researchers working in the area of porcine pluripotent cells. Within two years after the publication of the first papers describing human and mouse iPSCs, three different groups reported the establishment of iPSCs in the pig. One of these groups generated porcine iPSCs using fetal fibroblasts from Danish Landrace pigs, by the lentiviral transduction of six human transcription factors (OCT4, SOX2, c-Myc, KLF4, LIN28, and NANOG) under the control of a doxycycline-inducible promoter [41]. Simultaneously, another Chinese group reported the derivation of iPSCs from fibroblasts from the Tibetan minipig, using constitutively expressed lentiviral vectors carrying the mouse cDNA sequences of Oct4, Sox2, c-myc, and Klf4 [42]. The third report published shortly thereafter by Ezashi et al. [43] also described the production of porcine iPSCs through the use of 4 human transcription factors (OCT4, SOX2, c-Myc, and KLF4). In addition, these researchers claimed that the porcine iPSCs were positive for SSEA-1 but negative for SSEA3 and SSEA-4, indicating that they were more similar to mouse than human iPSCs.

the ultimate proof of pluripotency, and has been demonstrated only in the mouse and chicken to date. Human ESCs are presumed to be true stem cells as well, even though their germline potential has not been tested for ethical reasons. Caution is needed when interpreting chimera experimental results. Proper controls are necessary to distinguish the "true" stem cells from somatic cells that can be integrated into the embryos after partial reprogramming by the

The reasons for the limited pluripotency of the porcine EGCs are currently unclear. It is possible that the tissue culture conditions are not sufficient for maintaining the pluripotency of the PGCs *in vitro*, similar to the problems that exist in the cultures of inner cell mass and epiblast cells. Another possibility is that the pig PGCs do not undergo full epigenetic reprogramming into pluripotency like the mouse PGCs do. One indication that there might be differences in the biology of PGCs between the pig and mouse is that porcine PGCs survive and proliferate *in vitro* in the absence of externally added growth factors, regardless of whether they are cultured in serum-supplemented or serum-free conditions, while mouse PGCs fail to survive in the absence of any of the three growth factors LIF, SCF, or bFGF. In addition, mouse PGCs have not been shown to form chimeras, whereas porcine PGCs can contribute to somatic tissues after their injection into early blastocysts [24]. Thus, some of the molecular mechanisms that are important for reprogramming mouse PGCs to pluripotent EGCs in culture may be different

Takahashi and Yamanaka [37] discovered that the genome of a differentiated somatic cell can be epigenetically reprogrammed to pluripotency by the induced expression of pluripotency transcription factors, resulting in the generation of pluripotent stem cells [induced Pluripotent Stem Cells (iPSCs)]. These authors initially expressed 24 pluripotency genes in fetal fibroblasts to reprogram them into pluripotent cells, and subsequently found that the expression of only 4 of these genes, namely *Oct4, Sox2*, *c-myc*, and *Klf4*, was sufficient to achieve the same results. Nanog was dispensable for the cellular reprogramming. In addition, a year later, Okita et al. [38] produced germline-competent mouse iPSCs using the same growth factors, but by

The production of human iPSCs started shortly after the publication of the first report of mouse iPSC derivation. Thus, it was not surprising that the establishment of the first human iPSCs was reported by two groups simultaneously. The laboratory that produced the first mouse iPSCs successfully generated iPSCs from adult human fibroblasts by using the same 4 transcription factors employed in the initial experiment [39]. The other group, from the laboratory of human ESC pioneer James Thompson, was able to reprogram human fibroblasts

Predictably, these studies sparked significant interest among researchers working in the area of porcine pluripotent cells. Within two years after the publication of the first papers describing

surrounding embryonic cells.

202 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

**4. Induced pluripotent stem cells**

selecting the reprogrammed cells by Nanog expression.

by expressing OCT4, SOX2, NANOG, and LIN28 [40].

in the pig.

Transcriptional profiling of the cell line by Affymetrix microarray confirmed that the cells were indeed reprogrammed, and expressed a variety of ESC markers endogenously. However, the continued expression of the exogenous transcription factors was detected in the iPSCs generated by all three groups. This problem is not unique to the pig, as it has been reported in other species as well [39]. In any case, the continued expression of pluripotency genes did not pose any problems for the differentiation of the cells, since all three laboratories demonstrated that their cell lines were able to differentiate *in vitro* (including EB formation) and in vivo by forming teratomas containing cells of all three germ layers. More recently, it was demonstrated that porcine iPSCs can form chimeras with high efficiency (85.3%), and contribute to all three germ layers [44]. Because germline chimerism was not confirmed in this study, it remains to be determined whether the porcine iPSCs are fully equivalent to mouse iPSCs.

One of the major advantages iPSCs may offer in the future is the potential for custom derivation of pluripotent cells from individual patients to use for regenerative therapies without the risk of immune rejection. However, since the epigenetic reprogramming necessary to produce them requires prolonged expression of the transgenes (2-3 weeks), most of the iPSCs described to date have been generated with the use of lentiviral vectors that integrate known oncogenes (such as c-myc and Klf4) into the cell genome. The dangers of using these genes became evident in a study in which mouse chimeras generated with iPSCs developed tumors following reactivation of the initially silenced transgene c-myc [38]. In the only report to date in which porcine chimeras were produced from iPSCs [44], no tumor formation was reported in the newborn piglets. However, because long-term testing and monitoring were not conducted prior to the publication of these results, at present there is no guarantee that these iPSCs would be safe for clinical applications.

To avoid problems associated with viral integration and the use of oncogenes, many groups have developed strategies for iPSC production in which the disadvantages discussed above have been minimized. For example, it has been shown that reprogramming can be achieved in both human and mouse cells without the use of c-myc, albeit at the expense of efficiency [45]. In addition, the use of certain transcription factors can be omitted when using cell types already expressing them; for example, neural progenitor cells, which already express endogenous Sox2 and c-myc were reprogrammed using only the induced expression of OCT4 and Klf4 [46] [47]. Furthermore, some transcription factors can be replaced by small molecules that have similar effects on somatic cell reprogramming [48]. Viral integration, which carries risks of insertional mutagenesis, can be avoided by using nonintegrating adenoviral vectors [49] [50]. Our laboratory has established porcine iPSCs derived from pig skin fibroblasts using Sendai virus vectors to introduce 4 human transcription factors (OCT4, SOX2, c-Myc, and KLF4) (data not shown). Another attractive alternative to the use of viral vectors are transposon vectors, which combine high transfection efficiency with enhanced safety, and can be removed from the cell genomes following reprogramming. For example, piggyBac is a DNA transposon that, following insertion, can be removed from the reprogrammed genome without leaving a trace. This transposon has already been used successfully to produce iPSCs [51, 52]. Finally, Zhou [53] produced iPSCs by providing the transcription factors in the form of recombinant proteins.

**6. Conclusions**

cell research has a promising future.

and Technology of Japan, 2011-2015.

\*Address all correspondence to: hideyukk@med.toho-u.ac.jp

Department of Urology, Toho University School of Medicine, Tokyo, Japan

**Acknowledgements**

**Author details**

Hideyuki Kobayashi\*

**References**

1990;41:51-6.

865-77.

1990;34(5):879-901.

Stem cell biology research of the minipig is developing rapidly. Although studies on mouse and human stem cells currently outnumber those of other species, data from these well-studied species provide a good foundation for current and future porcine stem cell research. Despite the challenges associated with developing porcine pluripotent stem cells, recent successes in the fields of induced pluripotent stem cells and somatic stem cells suggest that minipig stem

The Minipig — A New Tool in Stem Cell Research

http://dx.doi.org/10.5772/57603

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This study was supported in part by a Grant-in-Aid for Young Scientists (B) of the Japan Society for the Promotion of Science (JSPS) and Grant of Strategic Research Foundation Grant-aided Project for Private schools at Heisei 23th from Ministry of Education, Culture, Sports, Science

, Toshihiro Tai, Koichi Nagao and Koichi Nakajima

[1] Notarianni E, Laurie S, Moor RM, Evans MJ. Maintenance and differentiation in cul‐ ture of pluripotential embryonic cell lines from pig blastocysts. J Reprod Fertil Suppl.

[2] Piedrahita JA, Anderson GB, Bondurant RH. On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos. Theriogenology.

[3] Piedrahita JA, Anderson GB, Bondurant RH. Influence of feeder layer type on the ef‐ ficiency of isolation of porcine embryo-derived cell lines. Theriogenology. 1990;34(5):

With regard to future research in porcine iPSC production, the choice of cell type and strategy will undoubtedly play an important role in the reprogramming efficiency. It should be noted, however, that the same problems existing in porcine ESC culture are likely to affect the longterm maintenance of the newly generated pig iPSC lines. For example, the culture conditions supporting the pluripotency and self-renewal of the porcine iPSCs apparently require further optimization, given that, in all the published reports to date, the reprogrammed cell lines could be maintained only with the continued expression of exogenous pluripotency genes. In one study, for example, instead of using growth factors or other supplements, the pig iPSCs were maintained with doxycycline-induced expression of the pluripotency transgenes, until the authors chose to differentiate the cells [41].

Despite these challenges, we can without doubt look forward to an exciting future in which iPSCs will add another dimension to pluripotent stem cell research in the pig.
