**The Minipig — A New Tool in Stem Cell Research**

Hideyuki Kobayashi, Toshihiro Tai, Koichi Nagao and Koichi Nakajima

Additional information is available at the end of the chapter

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

**1. Introduction**

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

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2004-15.

The establishment of pluripotent mouse embryonic stem cells (ESCs), which have the capacity to differentiate into all cell types in the mammalian body, was first described by Evans and Kaufman in 1981. Seventeen years later, in 1998, the first report of human ESCs, with similar differentiation characteristics was published by James Thompson. In 2006, Takahashi and Yamanaka discovered that the genome of a differentiated somatic cell could be epigenetically reprogrammed to pluripotency by inducing the expression of pluripotency transcription factors, resulting in the production of pluripotent stem cells (iPSCs). This research has accelerated the study of regenerative medicine.

In 2009, the US Food and Drug Administration approved the first Phase I clinical trials of human ESC-derived cells for the treatment of spinal cord injuries. However, previous reports had revealed that stem cell-based therapies increase the risk of tumor development. Therefore, further basic research is needed before stem cell-based therapies can be applied in the clinic. The development of stem cell tools can be critically evaluated using animal models that express human disease genes.

Research on the stem cell biology of the minipig is developing rapidly. Although research on mouse and human stem cells currently predominates over that in other species, data from these well-studied species have provided a good foundation for current and future porcine stem cell research. In addition, the increasing popularity of alternative-species models for the study of human diseases and disease mechanisms has further spurred porcine stem cell research. As a model system for pluripotent embryonic stem cell research, however, the pig presents several challenges as compared with mice and humans. Nonetheless, porcine minipig embryonic germ cells have recently been produced, and may prove particularly useful for *in vitro* and *in vivo* differentiation studies, gene targeting, and the creation of transgenic animals. In addition,

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studies involving the transplantation of somatic mesenchymal stem cells into porcine heart, cartilage, and bone have yielded very promising results. Lastly, minipig induced pluripotent stem cells have been established by using Sendai viruses to introduce pluripotency transcrip‐ tion factors into the cells.

by somatic cell nuclear transfer, using minipig fetal or neonatal fibroblasts as the donor and prepubertal gilt oocytes, followed by culture *in vitro* to the blastocyst stage of development. Two cells lines could be cultured for more than 48 passages, and expressed alkaline phospha‐ tase (AP), SSEA1 and SSEA3, OCT4, TRA-1-60, and TRA-1-81. The only method used to characterize their differentiation ability was the observation of spontaneous differentiation into embryoid bodies (EBs), which are spheres of cells that contain cell types of all three germ layers. These EBs were assessed solely by gene analysis, thus further characterization would

The Minipig — A New Tool in Stem Cell Research

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

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Only two reports to date describe the production of chimeras from porcine ESC-like cells: one in 1999, from Chen and colleagues [12], and a more recent publication in 2010 [13]. The first report claimed that somatic chimeric piglets could be produced, although clear analyses of these chimeric animals was lacking. The second report indicated that porcine ESC-like cells from an early passage could form chimeric piglets. The chimeric contribution was low, however; only 4 chimeric piglets were born after the transfer of hundreds of embryos. Only 2 of the 4 chimeric piglets showed coat chimerism, and this contribution was low and restricted to a single spot near the tail. Such a chimeric contribution is much lower than what would be expected from mouse ESCs, indicating that improvements to the cell culture conditions may

There are several possible reasons for the difficulty in producing porcine ESCs. The lack of defined culture conditions may be one reason. Pluripotency appears to be controlled by more than one cell-signaling pathway, and these pathways are different in mouse and human ESC lines. The origin of the cells, that is, the inner cell mass (mouse) versus the epiblasts (human) may contribute to this diversity of regulatory pathways. This idea is supported by a recent publication showing that even mouse EpiSCs regulate pluripotency slightly differently from human ESCs [14]. The cell signaling that governs pluripotency in the pig remains largely unknown, although the details are beginning to be investigated [15]. For example, it was reported that fibroblast growth factor (FGF) signaling may be active in porcine epiblasts, and that the JAK/STAT pathway is inactive. The Activin/Nodal pathway also appears to be active in porcine epiblasts [8]. Culturing porcine epiblasts in medium containing basic FGF (bFGF) cannot prevent their differentiation, indicating that other factors are apparently necessary to

Differences occurring during early embryonic development in the domestic pig, as compared with mouse and human, could also account for the observed difficulties in producing porcine ESCs. The early development of the porcine embryo prior to implantation takes longer and is less advanced than in the other species. The inner cell mass differentiates into the hypoblast and epiblast at a later time point than in the mouse and human, and the porcine epiblast expands and develops over a period of several days. The cell signaling controlling this development in the pig could differ markedly from that in the mouse or human, and should be investigated to ascertain which stage of development is optional for isolating the pluripotent cells. It is possible that the later epiblast is already predetermined at the cell-signaling level to undergo gastrulation, or that the inner cell mass cells have not yet acquired the necessary cell

be necessary before they could be verified as true ESCs.

be required to improve the plasticity of these cells.

help maintain cellular pluripotency.

signaling machinery to support proliferation.

Thus, despite the challenges of developing porcine pluripotent stem cells, recent successes in the fields of both induced pluripotent stem cells and somatic stem cells suggest that the future of research using minipig stem cells is quite promising.
