**6. Conclusions**

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.

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

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

Despite these challenges, we can without doubt look forward to an exciting future in which

Epiblast stem cells are pluripotent stem cells derived from the epiblast layer of post-implan‐ tation mouse embryos [54]. Epiblast stem cells are distinct from ES cells, which are derived from the inner cell mass of blastocysts. Both ES cells and epiblast stem cells can differentiate into mesoderm, endoderm, and ectoderm. However, epiblast stem cells do not have germ-line

Rodent pluripotent stem cells are considered to have two distinct states: naïve and primed. Naïve pluripotent stem cell lines are distinguished from primed cells in their response to LIF signaling and MEK/GSK3 inhibition (LIF/2i conditions) and X chromosome activation. Human ES and iPS cells both resemble rodent primed epiblast stem cells more closely than rodent naïve ES cells. In addition, iPS cells derived from pigs can obtain the properties of primed epiblast stem cells. iPS cells derived from human, monkey, rabbit, and rat, but not mouse, can

iPSCs will add another dimension to pluripotent stem cell research in the pig.

also obtain the properties of primed epiblast stem cells [55].

authors chose to differentiate the cells [41].

**5. Epiblast stem cells**

transmission.

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 cell research has a promising future.
