**2. Embryonic stem cells**

Most porcine embryonic stem cell research has been performed in large domestic pig breeds rather than minipigs. Therefore, this discussion will focus on studies performed on the large domestic breeds, yet with attention paid to outcomes observed in the minipig. Research in the domestic species is highly transferrable to the minipig.

Although researchers have sought to establish ESCs in the pig, the characterization of ESCs in this animal falls short, due to a lack of both *in vivo* and long-term culture studies as compared to ESCs of mouse or human origin. The first reports on ESC production in the pig were in 1990 [1] [2] [3] [4]. However, those attempts produced only putative ESCs or embryonic stem-like cells.

A number of standard techniques are typically employed to verify the identity of true ESCs. Several *in vitro* techniques are generally performed to determine the expression profile of the ESCs, including gene-expression and protein analyses. The transcription factors expressed in both mouse and human ESCs include OCT4, NANOG, and SOX2. In addition, stage-specific embryonic antigen 1 (SSEA1) is expressed in mouse but not human ESCs, and SSEA3 and SSEA4 are detected in human but not mouse ESCs [5].

While the efforts to establish porcine ESCs have been well reviewed in the scientific literature [6] [7], a general overview of the research suggests that true porcine ESCs have not yet been produced. The production of porcine epiblast stem cells (EpiSCs) was recently reported [8]. These cells are thought to be derived from the epiblast, rather than the inner cell mass, of the developing embryo. Evidence suggests that mouse ESCs are of inner cell mass origin, while human ESCs originate from epiblasts [9]. The porcine epiblast stem cells could be cultured for 22 passages, and could differentiate *in vitro* into cell types representative of the three embryonic germ lineages as well as germ precursor cells and trophectoderm. However, it is not known whether these cells also demonstrate pluripotency.

To date, two research groups have attempted to produce porcine ESCs from the minipig [10, 11]. Li and colleagues reported that outgrowth cultures could be obtained after isolating the inner cell mass from Chinese minipig blastocysts, although sustained culture was difficult to achieve, and only a preliminary characterization of these cells was performed [10]. Long-term cultures of porcine ESC-like cells were reported by Kim and colleagues [11]. In this study, porcine ESC-like cells were derived from cloned blastocysts. These embryos were produced 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 be necessary before they could be verified as true ESCs.

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‐

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

Most porcine embryonic stem cell research has been performed in large domestic pig breeds rather than minipigs. Therefore, this discussion will focus on studies performed on the large domestic breeds, yet with attention paid to outcomes observed in the minipig. Research in the

Although researchers have sought to establish ESCs in the pig, the characterization of ESCs in this animal falls short, due to a lack of both *in vivo* and long-term culture studies as compared to ESCs of mouse or human origin. The first reports on ESC production in the pig were in 1990 [1] [2] [3] [4]. However, those attempts produced only putative ESCs or embryonic stem-like

A number of standard techniques are typically employed to verify the identity of true ESCs. Several *in vitro* techniques are generally performed to determine the expression profile of the ESCs, including gene-expression and protein analyses. The transcription factors expressed in both mouse and human ESCs include OCT4, NANOG, and SOX2. In addition, stage-specific embryonic antigen 1 (SSEA1) is expressed in mouse but not human ESCs, and SSEA3 and

While the efforts to establish porcine ESCs have been well reviewed in the scientific literature [6] [7], a general overview of the research suggests that true porcine ESCs have not yet been produced. The production of porcine epiblast stem cells (EpiSCs) was recently reported [8]. These cells are thought to be derived from the epiblast, rather than the inner cell mass, of the developing embryo. Evidence suggests that mouse ESCs are of inner cell mass origin, while human ESCs originate from epiblasts [9]. The porcine epiblast stem cells could be cultured for 22 passages, and could differentiate *in vitro* into cell types representative of the three embryonic germ lineages as well as germ precursor cells and trophectoderm. However, it is not known

To date, two research groups have attempted to produce porcine ESCs from the minipig [10, 11]. Li and colleagues reported that outgrowth cultures could be obtained after isolating the inner cell mass from Chinese minipig blastocysts, although sustained culture was difficult to achieve, and only a preliminary characterization of these cells was performed [10]. Long-term cultures of porcine ESC-like cells were reported by Kim and colleagues [11]. In this study, porcine ESC-like cells were derived from cloned blastocysts. These embryos were produced

tion factors into the cells.

**2. Embryonic stem cells**

cells.

of research using minipig stem cells is quite promising.

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

domestic species is highly transferrable to the minipig.

SSEA4 are detected in human but not mouse ESCs [5].

whether these cells also demonstrate pluripotency.

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 be required to improve the plasticity of these cells.

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 help maintain cellular pluripotency.

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 signaling machinery to support proliferation.

We have yet to observe *bona fide* ESCs produced from the pig. Nevertheless, continued research toward this goal is important, as the pig is a particularly useful biomedical model for studying human disease, and ESCs are a unique cell type that is especially useful for studying human disease.

mouse fibroblast cells from a cell line known as STO. The initial establishment of cell lines is hampered by the fact that large numbers of porcine PGCs undergo apoptosis and die in culture within 6 hours of their incubation [25]. However, by using protease inhibitors and antioxidants to reduce the level of PGC apoptosis *in vitro*, Lee et al. were able to increase the number of EGC colonies in the primary culture. In addition, alpha 2-macroglobulin together with N-acetyl‐ cystein and butylated hydroxyanisole (both antioxidants) increased the number of AP-positive colonies in primary cultures at least twofold. Another approach is to use growth factors and various feeder cells to increase the survival and prevent the differentiation of PGCs in longterm culture. Lee et al. [26] showed that supplementing the culture medium with three growth factors (LIF, SCF, and bFGF) increased the number of colonies obtained in primary culture, and improved the quality of the colonies in subsequent passages compared to cells treated with only two of these growth factors. The use of feeder layers other than STO cells did not have a significant effect on PGC survival or on the quality of the derived EGCs. In another study, membrane-bound SCF and soluble LIF were sufficient to increase the number of surviving PGCs on days 3 and 5 of primary culture, while adding bFGF did not affect the results significantly [23]. It is possible that the membrane-bound form of SCF is more potent than the soluble form, which could explain these differing results. However, it has also been reported that established PGCs are capable of generating chimeras without added growth factors, while adding LIF to the culture does not improve the efficiency of establishing EGC lines [22]. Thus, further study is needed to elucidate the effects of these supplements in culture. Pluripotent stem cells are able to form EBs, spherical aggregates containing differentiated cells, when cultured in suspension. Porcine EGCs cultured in "hanging drops" can also form simple EBs, consisting of large epithelial-like cells on the periphery surrounding mesenchy‐ mal-like cells in the center. When allowed to attach to gelatin-treated plastic dishes, the EB cells proliferate and spread on the surface of the dish, giving rise to several different types

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Pluripotent cells can proliferate and differentiate *in vivo*, forming tumors containing differen‐ tiated tissues called teratomas, when injected into immunodeficient mice. To date, only one group has reported teratoma testing for porcine EGCs, using cell lines derived from the Chinese minipig [28]. The authors reported that the teratomas contained cells from the three primary germ layers: epithelial, neuroepithelial, and adipose tissue. In contrast to these results, two studies showed that the injection of human EGCs into immunodeficient mice failed to generate teratomas. [31] [32]. However, more recently, the formation of teratomas from human

Another way of testing the differentiation potential of pluripotent cells *in vivo* is by chimera formation, in which the cells injected into early embryos contribute to the three germ layers and potentially to the germline. Unlike mouse ESCs and EGCs, which have been shown to reenter the germline of chimeras, pig EGC chimeras have not displayed a proven germline contribution; furthermore, the somatic tissues of these porcine chimeras contained only a low percentage of donor-derived cells [22] [34] [24]. Other researchers demonstrated a similarly low chimeric contribution after the injection of somatic cells into sheep blastocysts and 8-cell mouse embryos [35, 36]. This is troubling because germline contribution is considered to be

EGCs cultured under serum-free conditions was reported [33].

of cells [27].
