Journal of Reproduction and Development, Vol. 53, No. 6, 2007, 19108

—Review—

Putative Embryonic Stem Cell Lines from Pig Embryos

Irena VACKOVA1,2), Andrea UNGROVA1) and Federica LOPES3)

1)Institute of Animal Science, Pratelstvi 815, 104 00 Praha Uhrineves, Prague, 2)Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic and 3)Dipartimento di Scienze Biomediche Comparate, Teramo University, Piazza Aldo Moro 45, 64100 Teramo, Italy

Abstract. Embryonic stem cells (ES cells) were first established in the mouse, and they represent apopulation of pluripotent, undifferentiated cells derived from early embryos that is capable ofproliferating without any limitation in an undifferentiated state. These cells retain the ability todifferentiate in vitro or in vivo into derivates of all three germ layers, and when injected intoblastocysts, they can participate in the formation of all tissues, including gonads (germ-line chimeras).It is possible to transfect them with a gene of interest, and the resulting transgenic cell lines can also beused for production of chimeras. Unfortunately, mammalian germ-line chimeras that can carry aninserted gene into their progeny have only been produced in the mouse. Logically, before applicationof stem cell therapies into a human medicine, it is necessary to verify the efficiency and safety of thesemethods with an acceptable animal model. The pig is currently used as a very convenient animal forpre-clinical applications, and therefore establishment of porcine ES cell lines is highly needed;unfortunately, no convincing ES cell lines have been produced in this species (and other domesticanimals) to date. In this article, we discuss the recent advances in this field, especially oriented onpossible reasons and obstacles why derivation of porcine ES cell lines is still unsuccessful.Key words: Chimera, Embryonic stem cells, Porcine

(J. Reprod. Dev. 53: 1137–1149, 2007)

he successful isolation and characterization ofmouse ES cells has resulted in efforts aimed at

establishing ES cell lines in other species, includinghamsters [1], minks [2], rabbits [3], rats [4], pigs [5,6], cattle [7], sheep [8], goats [9], horses [10] and innon-human primates [11] and humans [12]. Up tonow, these efforts have only been successful in pri-mates (including humans).

Logically, considerable effort has been exerted toestablish porcine embryo derived stem cell linesdue to the obvious physiological and immunologi-cal similarities between humans and pigs andespecially with the vision of potential practical use

in testing the safety of related technologies. In spiteof huge progress in the human ES cell field, it isabsolutely essential to investigate the therapeuticpotential of these cells with a model other than themouse before application of stem cell related thera-pies in humans.

It is commonly accepted that the substantial dif-ferences in early embryonic development betweenmice and other species are the limiting factor(s) pre-venting success of ES cell line establishmentstrategies [13]. Although the pattern of embryonicdevelopment from fertilization up to blastocyststage is rather similar in mammals, later on andespecially at the time of implantation, substantialdifferences can be detected. In the mouse, implan-tation is an invasive and rapid process. On the

Accepted for publication: September 13, 2007Correspondence: I. Vackova(e-mail: ivackova@seznam.cz, vackova@vuzv.cz)

VACKOVA et al.1138

other hand, in most ungulates, including pigs,implantation occurs only after a considerable delayduring which the trophectoderm proliferates rap-idly and the inner cell mass (ICM) forms aquiescent embryonic disc [14].

In our review, we are thus oriented especially onthe progress in derivation of embryonic stem celllines in the pig, the factors affecting their successfulestablishment, propagation and culture and ontheir characteristics and the possibility to differenti-ate them in vitro and in vivo.

The Age and the Source of Embryos

ES cell lines are conventionally isolated from theinner cell mass of blastocysts and eventually (butrather exceptionally) from earlier embryonic stages,i.e., single blastomeres [15], precompactionembryos [16] or morulae [17], and recently, pregas-trula stage embryos [18]. To our knowledge, pigES-like lines have only been established from blas-tocysts. There have been several exceptional, yetunsuccessful, attempts at derivation from 4- and 8-cell stage embryos and morulae produced in vitro[19]; however, the attachment rates of these pre-blastocyst stage embryos were low, and none of theembryos began outgrowth. Chen et al. [20] werealso unable to derive undifferentiated cell linesfrom porcine zona-free morulae, in spite of the factthat morulae formed flattened primary coloniesafter seeding under an STO feeder layer. Typically,as mentioned above, blastocysts at different stagesof development are used for derivation of porcineES-like cell lines. Day-5–6 postestrus in vivo blasto-cysts were used by Anderson et al. [21], Hocheraude Reviers and Perreau [20], Chen et al. [20] andShiue et al. [23].

Anderson et al. [21] attempted to assess whetherporcine blastocysts contain undifferentiated cellsthat are capable of further multiplication. ICMsfrom day-6 blastocysts did not survive the first pas-sage, and only 2% of them began outgrowth.Nevertheless, his group was able to establish ES-like cultures using ICMs from older blastocysts(d7–d10). The authors assumed that the factor(s)that may affect survival in culture was the size ofthe isolated ICMs; ICMs with fewer cells had areduced chance of survival. Chen et al. [20] wereunable to isolate a stable cell line from pre-hatchingblastocysts; colonies from early blastocysts grew

slowly after passaging and all consequently degen-erated, and those from expanding blastocystsproduced only differentiated cells that were all lostby passage 8. Hatched blastocysts older than 9 or10 days that have already begun to elongate or arealready elongated have been found to be unsuitablefor establishment of ES cells due to their fast differ-entiation in culture [20–22, 24, 25]. This finding wasconfirmed by Prelle et al. [13], who reported that theporcine embryonic disc begins to differentiate intomesodermal cells around day 9 (d9). On d9, about50% of evaluated embryos expressed intermediatefilament protein vimentin. On d7 and d8, vimentinwas found neither in the trophectoderm nor theICM, indicating that the porcine ICM is still pluri-potent at this stage. As development proceeds, theepiblasts of ovoid and tubular d10 and d11embryos showed an intense vimentin staining in allcases. These results suggest that, in the pig, d9 isthe latest stage at which undifferentiated cells arestill available in the ICM, and thus it is recom-mended that older blastocysts not be used.However, these observations are not in agreementwith those published by Strojek et al. [26], who com-pared d9 and d10 blastocysts and did not find anyoutgrowth in the culture for d9 blastocysts,whereas d10 blastocysts formed colonies of ES-likecells. Generally, most authors have used expandedor early-hatched blastocysts [6, 20, 23, 24, 27–32].This essentially corresponds with the results ofChen et al. [20], indicating that in the pig, an obvi-ous increase in the size of the ICM begins duringthe early-hatched blastocyst stage. Thereafter, pro-liferation of this cell population slows down andreaches a more quiescent state, whilst the trophec-toderm remains mitotically active.

In the pig as well as in other farm animals, themajority of authors have used in vivo producedblastocysts from superovulated animals. Eventhough in vivo produced embryos are expected tobe of very high quality, this approach is expensiveand laborious. Thus, it would be desirable, for eco-nomic reasons, to use embryos produced from invitro matured and fertilized oocytes. However, upto now, only a few ES-like lines have been estab-lished in farm animals from in vitro producedmaterial [33–35]. The first attempt to use in vitroblastocysts in the pig was described by Miyoshi etal. [36]. Their results indicate that a porcine cell linecan be isolated from an in vitro produced hatchedblastocyst and maintained in vitro for a relatively

1139REVIEW: PORCINE EMBRYONIC STEM CELLS

long period. Similarly, Li et al. [19] used IVFderived porcine embryos for the production of ES-like lines. They were able to maintain these cells inculture only from one blastocyst and maximallyonly for three passages. In general, it is very diffi-cult to isolate ICMs from in vitro producedblastocysts because in most cases there is no obvi-ous ICM or they eventually contain only few cells.This is also our experience. Nevertheless, we havesuccessfully established embryo-derived lines fromone IVF and one ICSI produced blastocyst (Vack-ova, unpublished). Ock et al. [37] compared theeffectiveness of IVF, parthenogenetically activated,PA, and in vivo-produced porcine embryos forestablishment of ES cell lines. Attachment rates ofICMs onto STO feeder layers were highest for invivo embryos followed by PA and IVF embryos.Nevertheless, formation of primary colonies waslow in all cases, and just one ES-like line was estab-lished from in vivo derived blastocyst. Brevini et al.[38] also used porcine PA blastocysts, but in con-trast to Ock et al. [37], they were able to establishputative embryonic stem cell lines expressing Oct-4and nanog. Kim et al. [39] compared the abilities ofIVF, somatic cell nuclear transferred, SCNT and PAblastocysts to form primary colonies. The IVF andSCNT groups formed more primary colonies thanthe PA group. Unfortunately, most of the coloniesmaintained their characteristics as ES cells untilthree to four passages and then differentiated ordegenerated. Up to now, parthenogenetic embry-onic stem cell lines have only been established inmice [40], non-human primates [41] and humans[42].

The use of frozen-thawed (FT) and ICSI pro-duced blastocysts for production of porcine stemcell lines has yet to be reported in spite of the factthat the above techniques have been used (with andwithout success) for production ES cell lines inother species (horse FT [10]; sheep FT [35]; primateICSI [43]; human ICSI [44]; human FT [45]).Another possibility is to establish ES cell lines fromblastocysts produced by nuclear transfer. In the-ory, this approach means production of patient-tailored (compatible) ES cells. However, thisapproach has only been successful in mice [46] andcattle [34] and has failed in porcine [20, 39].

ICM Isolation

Whole intact blastocysts without the zona pellu-cida or isolated ICMs can be used for establishmentof ES cell lines. Intact porcine blastocysts wereused by Evans et al. [14], Piedrahita et al. [6],Hocherau de Reviers and Perreau [22], Gerfen andWheeler [27], Miyoshi et al. [36], Li et al. [19] andKim et al. [39]. On the other hand, Talbot et al. [47]does not recommend culture of intact pig blasto-cysts because this approach results in attachmentand growth of the trophoblast and primitive endo-derm, whilst the embryoblast typically does notgrow and instead degenerates or differentiates.They and others [14, 22] suggest that successfulestablishment of porcine stem cell lines requiresisolation of the epiblast from the trophectodermand primitive endoderm. However, it is still diffi-cult to isolate ICMs from in vitro producedblastocysts due to not so prominent ICMs andICMs containing only a few cells [19, 48]. The com-monly used method for isolation of ICMs free of thetrophectodermal cells is immunosurgery [49]. Thisapproach has also been successfully used in the pig[6, 20, 21, 23, 28, 29, 37, 38]. Wianny et al. [25] devel-oped another immunosurgical method when usinga monoclonal antibody SN1/38 specifically raisedagainst porcine trophectodermal cells [50]. Othermethods used include treatment with calcium iono-phore [51], microsurgery [22, 24, 32, 48] and adigestive method using 0.25% Trypsin/EDTA [30,31].

Talbot et al. [47] suggested that pig ICM is com-posed of an inner core (epiblast) surrounded bymultiple layers of outer cells. The outer layers seemto be the primitive endoderm because in culturethey can only give rise to vesicles and dome-form-ing cells. They reported that immunosurgeryremoved most or eventually all of the trophectoder-mal cells but little or none of the endodermal layerforming the outer layer of the ICMs. The innermostlayer of endoderm is closely adhered to the epiblastcore, and it can only be removed after two days inculture. Hence, Talbot et al. [47, 52, 53, 54] recom-mended a 3-step dissection protocol consisting ofimmunosurgery followed by short culture and sub-sequent physical dissection of the ICMs from therapidly growing endodermal layer. This leads toisolation of the epiblast not only from the tropho-blast but also from the endoderm.

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Culture Conditions

Maintenance of porcine ES cells in culture is stilldifficult and suboptimal because of the totalabsence of suitable culture conditions capable ofinhibiting differentiation of isolated ICMs. The cul-ture systems that are generally used are based onmouse ES cell culture methods [55] and are notoptimal for porcine ES cell line derivation. Themost probable reason is that the mouse has a shortinvasive preimplantation period, while the pigexhibits a longer non-invasive preimplantationperiod that is characterized by rapidly dividing tro-phoblastic cells and a rather quiescent state in theICM [29]. Nevertheless, most laboratories use aculture system similar to the mouse method, con-sisting of DMEM supplemented with L-glutamine,2-mercapthoethanol, antibiotics, nucleosides, non-essential amino acids and different sources anddoses of serum [5, 6, 14, 19–31, 37, 47, 51].

Moore and Piedrahita [29] compared a com-monly used murine ES cell medium (DMEM withhigh glucose = D medium) with a commonly usedmedia used in culture of livestock embryos (DMEMwith low glucose and Ham’s-F10 1:1=D/Hmedium). Their results demonstrate that D/Hmedium might be superior compared with thecommonly used D medium, especially when estab-lishing long-term cultures of porcine embryoniccell lines. Low glucose DMEM/F10 medium hasalso been successfully used for establishment of EScell lines from PA blastocysts [38] and is superior toDMEM alone [39].

Maintenance of mouse ES cells depends either onthe presence of a feeder layer that is capable of syn-thesizing and secreting leukemia inhibitory factor(LIF) or on exogenous supplementation with puri-fied LIF protein and eventually on the use ofconditioned media [29]. Previous reports haveshown only little or no beneficial effect of additionof hLIF on the maintenance of porcine stem cells inan undifferentiated state [22, 25, 28, 29, 47, 52].Thus, it would be desirable to compare the effi-ciency of hLIF with the effect of homologousporcine LIF [52]. Some authors have attempted touse other cytokines and growth factors to improvethe culture conditions. It has been found that addi-tion of bovine insulin [22, 26], platelet-derivedgrowth factor, PDGF [22], basic fibroblast growthfactor, bFGF [19, 30–32, 36, 48, 56], Ewing sarcomagrowth factor, ESG [9], and Buffalo rat liver (BRL)

cell-conditioned medium [36] have favorableeffects on successful isolation and/or proliferationof porcine stem cells.

Some factors that were found to suppress the dif-ferentiation of mouse embryonic cells have nodifferentiation-inhibiting effect on porcine stemcells, including transforming growth factor beta(TGFβ), epidermal growth factor (EGF) [22], eryth-roid differentiation factor (hrEDF) [47], insulin-likegrowth factor 2 (IGF-2) [51], conditioned medium[24], interleukin 6 (hIL-6), oncostatin M (hOSM)and ciliary neurotrophic factor (rCNTF) [28].

According to Goldsborough et al. [57], fetalbovine serum is the main source of potential differ-entiating factors in ES cell cultures. To maintain EScells in an undifferentiated state, they recommendKNOCKOUTTM Serum Replacement (KO-SR;Gibco, Grand Island, NY, USA), a defined serum-free supplement, be used instead of animal serum.In addition, an optimised DMEM formulation,KNOCKOUT DMEM, can further improve ES cellcultures. KO-SR was used only rarely for isolationand maintenance of porcine embryonic stem celllines [32, 38, 48]. Other authors have used fetalbovine serum (FBS), fetal calf serum (FCS), new-born calf serum (NCS) or their combinations. OnlyStrojek et al. [26] compared the influence of serumsupplementation on the formation of morphologi-cally undifferentiated cells from porcine ICMoutgrowths. The viability of embryos and theirpotency to form ICM-derived colonies was highestwhen porcine serum was used in combination withfetal calf serum compared with calf serum only orhuman cord serum supplementation, respectively.

Because the typical expansion and elongation ofporcine blastocysts does not occur under in vitroculture conditions, Ropeter-Scharfenstein et al. [24]attempted to create a culture system that wouldallow the embryo to survive without differentiationup to the onset of ICM proliferation. To simulate invivo conditions, the embryos were cultured on agelatinous collagen matrix. Their two-stage culturesystem consisted of preculture in NCSU23-mediumcontaining insulin, transferrin and Na-selenite on athree-dimensional collagen matrix for 4 days withsubsequent culture in DMEM on an MEF feederlayer not inactivated by mitomycin. The embryosincreased to three times in size and appeared to beperfectly viable, and ICM cells taken from precul-tured embryos survived longer than control cells(up to 13 passages).

1141REVIEW: PORCINE EMBRYONIC STEM CELLS

The method of cell passaging is another impor-tant factor that is crucial for establishment andmaintenance of ES cells in culture. For this pur-pose, trypsin has been successfully used for murineES cells [58]. In other species, similar attempts haveled to loss of proliferation and/or to induction ofcell differentiation, including in cattle and rabbits[59], pigs [47], monkeys [43], and humans [60].However, the majority of researchers have reportedsuccessful trypsinization of porcine pluripotent EScells [14, 19, 21–23, 25–27, 30, 31, 36, 37, 51, 61] bytreatment with trypsin or trypsin/EDTA.

Feeder Cell Layer

The feeder cell layer is one of the most importantfactors affecting ES cell culture. It serves as anattachment matrix for cells and can secrete somekinds of cytokines, such as LIF, that may stimulateES cell growth and inhibit their differentiation [30].Proliferation of feeder cells must be prevented sothat they can be used as nonreplicating viable sup-porting cells. Mitomycin C and γ-irradiationtreatments are commonly used for inactivation offeeder cells. They inhibit DNA synthesis whileRNA and protein synthesis continues; the cellsremain vital, although they are unable to divide.Currently, the established cell lines from mouseembryonic fibroblasts STO and MEF and homolo-gous embryonic fibroblasts (HEF) are the mostcommonly used feeder cells.

Some experiments have been designed to analyzewhich feeder cells are the most suitable for porcineES cell cultures, but the results obtained have beenrather inconsistent. While Strojek et al. [26] pre-ferred a feeder layer from porcine uterine cells(PUC) and did not recommend STO cells. Piedrah-ita et al. [5, 6] found an STO feeder layer to be moresuitable because it supports the growth of embryoderived colonies for longer than 10 passages. Basedon testing of other feeder layers by Piedrahita et al.[5], PUC buffalo rat liver cells (BRL) and epithelial-like porcine embryo-derived cell line (PH3A) donot support attachment of the porcine ICM. Per-haps due to the reduced or missing extracellularmatrix in porcine embryonic fibroblasts, PEF doesnot allow for proliferation of attached cells, andMEF induces quick differentiation into a presump-tive endodermal or trophoblastic cell layer. Kim etal. [39] also did not observe any attachment of the

ICM in PUCs or under feeder-free conditions. Tal-bot et al. [47] suggested that culture of STO cellscontributes to differentiation of the pig epiblast.Hocherau de Reviers and Perreau [22] compared afeeder-free culture system with culture on a PEFfeeder layer. The longest duration of culture of ES-like cells with a morphology resembling mouse EScells was achieved on the PEF feeder layer. Accord-ing to Ropeter-Scharfenstein et al. [24], MEFs werethe best feeder for ES-like cells, with the highestnumber of passages obtained. The attachment rate,proliferation or viability of ICMs with the otherfeeders tested (PEF, neonatal goat testes, porcinetrophoblast vesicles, porcine granulosa cells andestablished cell lines, including LLCPK, 3T3, P19and BRL) was rather low. When Li et al. [31] com-pared three different feeders, they found that PEFproduced the same results as MEF, and both weresuperior to STO. The attachment rate and primarycolony formation on STO cells were significantlylower, and the majority of the cultured cells differ-entiated after the 1st passage. They also examinedmicrotubules in the feeder cells and found that themicrotubule localization of PEF was the same asthat of MEF; however the pattern was significantlydifferent from that in STO. Also, the number ofmicrotubules was higher in MEF and PEF com-pared with STO. The authors concluded that thiscould be the reason why STO was unsuitable as afeeder layer because cells with fewer microtubuleshave reduced secreting activity. Nevertheless,most porcine ES-like cells have been derived usingthe STO feeder [5, 6, 14, 20, 21, 27, 36–39, 48, 52, 56,61].

Characterization of Embryo DerivedPorcine Cells

MorphologyThe morphology of embryonic stem cells should

have two major traits: i) the smallest amount ofcytoplasm and large nucleus with one or two prom-inent nucleoli and ii) the fastest proliferation in thegiven population [59]. The slower proliferation rateof undifferentiated, stem-like cell explants of por-cine embryos compared with those derived frommurine embryos [6, 24] may reflect the above-men-t io n ed d i f f e r en c es in p r e - i m p l a n t a t i o n a ldevelopment between these two species. That is,the period of quiescence in the inner cell mass of

VACKOVA et al.1142

ungulates lasts up to the time of gastrulation [14].Due to morphological and developmental differ-ences between murine and porcine embryos, wecan also logically expect morphological differencesbetween corresponding colonies [2]. Nevertheless,the majority of porcine embryo-derived cells, as inthe mouse, have large, translucent nuclei, one ortwo prominent nucleoi and relatively little cyto-plasm [5, 14, 20–22, 25, 27, 47, 61, 62].

Some authors [19–21, 28–31, 36, 47, 61] haveobserved, typically soon after seeding onto a feederlayer, the presence of compact colonies growing astightly packed mounds and containing smallrounded cells resembling mouse ES cells. How-ever, maintenance of this morphology is difficultbecause porcine ICM cells quickly differentiate.

In general, the individual cells in porcineembryo-derived cell lines are morphology similarto mouse ES cells; however, the overall morphol-ogy of colonies is more epithelial-like [20]. Theepithelial-like colonies are usually characterized asround compact monolayers with clear colony bor-ders growing onto a feeder layer, sometimes dome-forming. The cells of these colonies have a highnucleocytoplasmic ratio. These epithelial-like cellsare flat, either cuboidal or eventually with polygo-nal or irregular in shape and size, with distinct cellborders and abundant lipid-like vacuoles [14, 19,20, 27–31, 36, 47]. Gerfen and Wheeler [27] noticedthe higher resistance of porcine embryonic cells totrypsin disaggregation. This might be an indicationof epithelial-like cell characteristics, since manyepithelial cell lines have been reported to be poorresponders to trypsin in relation to cell dispersion.

Scanning electron microscopy (SEM) of porcineES cells has confirmed the presence of rounded orpolygonal closely associated cells with numerousmicrovilli abundant on the apical cell surface [27,61]. SEM has also shown that undifferentiatedICMs are very compact, with no distinctive cellularjunctions [29]. These junctions become more appar-ent as differentiation proceeds. The advancingdifferentiation also results in increased celldiameter.

Immunocytochemistry and PCRMarkers specific either for undifferentiated or

differentiated cell types are commonly used forcharacterization of ES cell lines. The relative abun-dance of alkaline phosphatase (AP) is one of themost commonly used markers of the undifferenti-

ated pluripotent state of cells. AP labelling hasconsistently been found in the earliest developmen-tal stages of embryos and in undifferentiatedmouse ES and embryonal carcinoma (EC) cells [52],but this labelling is lost as soon as the cells differen-tiate. The same also seems to be true for porcinecells [19, 20, 29–31, 52]. The only exception is inneuron-like differentiated cell cultures that stillremain positive for AP [20, 52]. In a previous studyof porcine blastocysts, in contrast to the trophecto-derm, the ICM was uniformly and intensely stainedfor AP [52]. Positive staining was also detected inporcine embryo-derived cells from the earlieststages of culture up to the time of their differentia-tion. The intensity of AP staining in the endoderm-like colonies was weak and sporadic.

Another marker that has been shown to functionas a key transcription factor in the maintenance ofpluripoteny in early embryos and ES cells in themouse and human is the Oct-4 (POU5F1) transcrip-tion factor [63]. Surprisingly, Oct-4 protein waspresent in both the ICM and trophectoderm of notonly porcine in vivo and in vitro blastocysts [64], butalso in the trophectoderm of cattle and goats [64, 65,66]. Furthermore, Keefer et al. [65] detected Oct-4gene expression by RT-PCR in porcine endodermcell lines and caprine embryonic cell cultures anddetected it by immunocytochemistry in the tro-phectoderm of day 11 porcine ovoid blastocysts.Contrary to this, Vejlsted et al. [67], who examinedpost-hatching blastocysts 8–9 days postinsemina-tion, reported localization of Oct-4 exclusively inthe nuclei of ICMs. The only study that investi-gated the expression of Oct-4 in porcine ES-like cellcolonies was done by Brevini et al. [38]. RT-PCRrevealed Oct-4 positive colonies even after the 4th

passage. These colonies were also positive foranother marker of an undifferentiated phenotype,nanog. Further more, it is well documented thatmouse ES cells are positive for the surface antigenSSEA-1. A previous study showed that freshly dis-sected porcine ICMs also exhibited positiveimmunostaining for SSEA-1, whilst the surround-ing trophectodermal, and endodermal layer werenegative [25]. Nevertheless, SSEA-1 labelling wascompletely lost as early as when these cells weretrypsinized.

Intermediate filament protein vimentin, themarker of mesodermal differentiation, has not beenfound in porcine ES-like cells [6, 14, 24, 51]. Also,another marker of differentiation, cytokeratin 18

1143REVIEW: PORCINE EMBRYONIC STEM CELLS

(CK 18), which is a component of the cytoskeletonof differentiated polarized cells, has not beendetected in the porcine ICM [25, 29]. In contrast,several previous studies have shown that all thecells constituting the trophectodermal and endo-dermal layers exhibited strong immunoreactivity tocytokeratin and that the expression of CK 18 pro-gressively increased after culturing ICMs in vitro [6,25]. Expression of laminin, a factor of the extracel-lular matrix that is expressed in differentiatedendoderm-like cells, was not detected by Wianny etal. [25] in the freshly isolated ICM and trophecto-derm, but it was detected in the freshly isolatedprimitive endoderm layer and in ICM cells afterplating. Lamins A/C, which are not expressed inundifferentiated mouse and human ES cells [68],have been detected in porcine ES-like cells [69].

Spontaneous and Induced Differentiationof ES Ccells

In vitro differentiationThe ability of porcine embryo-derived cells to

differentiate in vitro was evaluated using three dif-ferent strategies: i) spontaneous differentiation, ii)ability to create embryoid bodies and iii) chemicallyinduced differentiation. Usually, when cells arepermitted to reach a high density in prolonged cul-ture, their differentiation occurs spontaneously [14,19–21, 27, 28, 30, 31, 47, 61]. They tend to differenti-ate into various cell types, including those ofmesodermal, neuroectodermal, ectodermal andendodermal origin. Muscle [14, 19, 21, 27, 30, 31,61], fibroblast [14, 19, 20, 27, 30, 31, 61], adipocyte[27, 61], macrophage [70], neural-like [14, 19, 20, 27,30, 31, 54, 61], endoderm-like [14, 20, 28], trophecto-derm-like [19, 20, 30, 31, 54] and epithelial-like celltypes [19–21, 27, 28, 30, 31, 61] have also beendescribed. Overtly differentiated cells normally failto re-attach and are lost during sub-culture [14, 27].The amount of lipid droplets decreases in differen-tiated colonies [19, 20, 30, 31], and the proliferationof colonies gradually decreases [20, 25]. The APactivity of differentiated cell types rapidly disap-pears [19, 20, 23, 29–31, 52], with the exception ofneural-like cells [20, 23, 52].

The possibility of chemically-induced differentia-t ion has been tested after addit ion of a l l -transretionic acid (RA) [20, 25, 61], or dimethyl sul-foxide (DMSO) [61], to culture medium in the

presence of a feeder layer. The ability to respond toRA and DMSO treatment by differentiation into awide range of cell types suggests that porcine ES-like cells are not yet terminally differentiated.

When cells were seeded onto non-adhesive sub-strates and cultured in suspension, they formedaggregates resembling mouse embryoid bodies(EB) [6, 14, 19, 20, 22, 23, 27, 30, 31, 38, 39, 61]. Themajority of these cystic embryoid bodies differenti-ated from epithelial (ectodermal) into endodermaland mesodermal cell types, which was confirmedby molecular analysis [38]. The differentiation wasmuch more extensive when EBs were permitted toattach to the new feeder layer [14, 20, 27, 38]. Ingeneral, the ability to form embryoid bodies thatcan give rise to different differentiated cells indi-cates that the colonies obtained from porcineembryos are at least pluripotent.

In vivo differentiationTo test their pluripotency, ES cells are injected i)

into nude mice and the formation of teratomas isexamined or ii) into blastocyst-stage embryos toproduce chimeras. The first attempt to analyze theeventual formation of teratomas in athymic micewas unsuccessful [6]. Nevertheless, later on whenHocherau de Reviers and Perreau [22] subcutane-ously injected nude athymic mice with porcine ES-like cells, teratomas composed of abnormal carti-lage mixed with blood cells and hematopoietictissue surrounded by muscles and epitheliaformed. On the other hand, no teratomas weredetected in a mouse that had received irradiatedporcine fibroblasts.

Up to now, chimeric offspring in the pig havebeen obtained from blastocysts injected with eitherfresh ICM cells [21, 71–73], cultured EG cells [74] orES cells [20, 61] or eventually from injections of EScells into the perivitelline spaces of 4- to 8-cell stageembryos [75]. Production of chimeric piglets eitherfrom blastomere aggregation [76] or morula injec-tion [72] has been unsuccessful. Germ-linechimerism in the pig has only been reported byAnderson et al. [21], Onishi et al. [72] and Nagash-ima et al. [73], but these authors used freshlyisolated ICM cells for injection into the blastocoel.

In spite of the intensive effort to produce chime-ras by injecting cultured porcine ES-like cells intorecipient blastocysts [6, 20, 21, 23, 27, 61, 75, 77], thebirth of chimeric piglets, documented by coat-colour chimerism and DNA markers, has only been

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demonstrated in three cases [20, 61, 75]. Notarianniet al. [77] reported chimerism and transgenesis inthe pig when using an embryonic cell line trans-formed by electroporation. Their results representthe first report of transgenesis in the pig at fetalstages, with the exception of the most commonlyused method, pronuclear injection. Golueke et al.[75] reported the birth of piglets after injection ofcells from transgenic an embryonic cell line into invivo embryos. Three piglets were confirmed posi-tive for the marker gene in various endoderm and/or mesoderm derived tissues, and the three remain-ing piglets had positive skin samples, suggestingcontribution to ectoderm derived tissues. How-ever, germ-line chimerism has not been confirmedin any cases [20, 61, 75, 77].

Chen et al. [20] analysed not only the in vivodevelopmental competence of injected embryos butalso the integration of ES-like cells into the ICMs inthe host embryos. FITC-labelled ES-like cells wereincorporated not only within the host ICM but alsowithin the trophectoderm of expanding blastocyststhat developed from injected early blastocysts. Theinfluence of passage number and duration of invitro culture on the ability of porcine ICM-derivedcells to produce chimeric embryos were deter-mined by Shiue et al. [23]. They found that culturedcells had the potency to integrate into both the ICMand trophoblast of the host blastocyst. They alsomentioned that prolonged culture for up to 6 daysin each passage has detrimental effects on chimerageneration.

The results of the above-mentioned papersdescribing ES cells derivation from pig embryos aresummarized in Table 1.

Conclusion

Establishment and maintenance of ES cell linesfrom porcine blastocysts is much more complicatedthan from mouse embryos or primate embryos. Todate, there are no suitable culture conditions forpreventing of the spontaneous differentiation andoccurrence of senescence in putative porcine ES celllines. Also, it is not yet clear which feeder layer isthe most convenient for porcine cells, either forestablishment of ES cell lines or maintenance ofthese cells in the undifferentiated pluripotent state.Two basic types of cell morphologies are observedin porcine embryo derived cultured cells, ES-like

and epithelial-like cells. Porcine embryo-derivedcells are similar to mouse ES cells; they have largetranslucent nuclei, one or two prominent nucleoliand a high nucleocytoplasmic ratio. They prolifer-ate more slowly than murine ES cells and have atendency to differentiate rapidly in in vitro culture.The ability to differentiate in vitro, either spontane-ously or after controlled induction, into differentcell types indicates that embryo-derived porcinestem cells are at least pluripotent. The in vivo differ-entiation potential of porcine ES cells has beenconfirmed by the formation of teratomas after sub-cutaneous injection of these cells into nude mice[22] and by creation of chimeric offspring after blas-tocyst injection [61]. Unfortunately, germ-linechimerism in pigs has not been confirmed. How-ever, in spite of great research activity, no ES cellsfrom any vertebrate species other than the mouse[58] and chicken [78] colonize the germ-line.

Embryonic stem cells are considered to be a pow-erful tool for therapeutic applications, includingtissue regenerative medicine and tissue engineer-ing in human medicine. Before these methods areintroduced into practice, it is logical that there is theevident requirement to verify their efficiency andsuitability and to exclude any detrimental effects.As the mouse physiology, anatomy and life spandiffer significantly from that of humans, the rodentmodels seem to be inappropriate [79]. Due toimmunological and physiological similaritiesbetween pigs and humans and due to a morediverse genetic background, the pig seems to bemuch better animal model compared with the com-monly used rodents. There is also a great interest inusing genetically modified pigs as organ donors forxenotransplantation, as well as a model for humandiseases [80].

Pluripotent cell lines can provide a suitable invitro model for studying early embryonic develop-ment and can be used for gene target ingtechnologies. Although a germ-line chimera hasyet to be obtained in the pig, nuclear transfer tech-nology with transfected ES-cells provides analternative option for gene targeting. Gene transferin pigs can thus be focused not only on establish-ment of suitable animal models for studyinghuman diseases and stem cell therapies but also forproduction of different tissues and organs forxenotransplantation. These approaches can alsolead to further improvement in productivity traits,reproductive performance and disease resistance in

1145REVIEW: PORCINE EMBRYONIC STEM CELLS

Tab

le 1

.Su

mm

ary

of im

port

ant

pape

rs d

escr

ibin

g d

eriv

atio

n of

por

cine

ES

cells

Aut

hors

Max

Age

and

Cu

ltiv

atio

nIC

MFe

eder

Mar

ker

In v

itro

In v

ivo

pass

age

sour

cem

ediu

mis

olat

ion

laye

rex

pres

sion

dif

fere

ntia

tion

plur

ipot

ency

num

ber

of e

mbr

yos

Eva

ns e

t al.,

199

0 [1

4]>1

yea

riv

7–9

dD

+ C

S, F

CS

inta

ct o

r is

olat

ed E

DST

OV

IME

B, m

uscl

e, n

eur,

fibr

obl,

end

oP

ied

rahi

ta e

t al.,

199

0 [5

, 6]

32 p

iv 7

–8 d

D +

FB

S, C

Sin

tact

or

imm

unos

urg

ery

dif

fere

nt k

ind

s of

feed

erV

IM, C

K18

ES-

like:

no,

Epi

thel

ial l

ike:

EB

no te

rato

mas

, no

chim

era

Stro

jek

et a

l., 1

990

[26]

5 p

iv 1

0–11

dD

+ d

iffe

rent

ser

aPU

C, S

TO

Hoc

hera

u d

e R

evie

rs &

8 w

ksiv

5–6

d o

r 10

–D

+ F

CS

(LIF

), d

iffe

rent

inta

ct o

r m

icro

surg

ery

feed

er fr

ee o

r PE

FE

B w

ith

dif

fere

ntia

ted

cel

lsd

evel

opm

ent o

f P

erre

au, 1

993

[22]

11 d

gro

wth

fact

ors

tera

tom

as in

nud

e m

ice

Tal

bot e

t al.,

199

3 [4

7, 5

2]80

piv

7–8

dD

or

D/M

199

+ FC

S, F

BS

3 st

ep d

isse

ctio

nST

OA

Pm

usc

le, n

eura

l, fi

brob

l, en

do,

ad

ipo,

ep

ithe

lA

nder

son

et a

l., 1

994

[21]

10 p

iv 6

–10

dD

+ F

BS,

CS

imm

unos

urge

ryST

Om

usc

le, e

pith

el, f

ibro

blno

chi

mer

aP

relle

el a

l., 1

994;

199

5 [5

1, 5

6]4

piv

8 d

D +

dif

fere

nt g

row

th fa

ctor

sV

IMca

lciu

m io

noph

ore

(bFG

F)

Whe

eler

, 199

4 [6

1]44

piv

bla

stoc

ysts

SC m

ediu

m th

en B

RL

-CM

STO

then

feed

er fr

eeE

B, m

uscl

e, fi

brob

l, ad

ipoc

yte,

neu

ral

coat

col

our

chim

eris

mG

erfe

n &

Whe

eler

, 199

5 [2

7]ce

ll lin

esiv

5.5

–7.5

dD

+ F

BS

inta

ctST

OE

B, m

uscl

e, n

eura

l, ad

ipoc

yte,

epi

thel

, no

chi

mer

afi

brob

lM

oore

& P

ied

rahi

ta, 1

996;

no p

assa

geiv

7 d

D o

r D

/H +

FB

S +

im

mun

osur

gery

gela

tini

zed

wel

lsA

P, C

K 8

.13

end

o, e

pith

el19

97 [2

8, 2

9]cy

toki

nes

Rop

eter

-Sch

arfe

nste

in e

t al.,

4 p

iv 7

–9 d

D +

dif

f. fa

ctor

s or

CM

mic

rosu

rger

yD

iffe

rent

kin

ds

ofV

IM, k

erat

in19

96 [2

4] fe

eder

Wia

nny

et a

l., 1

997

[25]

2 p

iv 7

or

11 d

D +

FC

S (L

IF)

imm

unos

urge

ry w

ith

PEF

SSE

A1,

CK

18,

spon

tane

ous

dif

fere

ntia

tion

, ind

uced

SN1/

38 o

r m

icro

surg

ery

lam

inin

dif

fere

ntia

tion

by

RA

trea

tmen

tN

otar

iann

i et a

l., 1

997

[77]

iv 8

dST

Och

imer

ism

and

tran

sgen

esis

at f

etal

sta

geG

olu

ecke

et a

l., 1

998

[75]

2 m

onth

siv

6–7

dtr

ansg

enic

chi

mer

ic p

igle

tsC

hen

et a

l., 1

999

[20]

35 p

iv m

orul

a or

6D

+ F

BS–

8in

tact

or

imm

unos

urg

ery

STO

AP

EB

, neu

ral,

fibr

obl,

end

o, T

E, e

pit

h,

chim

eric

pig

lets

, NT

d o

r N

Tm

usd

e- b

last

ocys

tM

iyos

hi e

t al.,

200

0 [3

6]30

pIV

F ha

tche

dN

CSU

-23,

BR

L-C

M

inta

ctST

O th

an g

elat

iniz

ed

NT

- d

evel

opm

ent t

o +

FC

S (b

FGF,

LIF

)w

ells

blas

tocy

st s

tage

Li e

t al.,

200

3; 2

004

[30,

31]

9 p

iv 7

–9 d

D +

FB

S (b

FGF,

LIF

)im

mun

osur

gery

, dig

esti

veM

EF,

PE

F, S

TO

AP

EB

, mus

cle,

neu

ral,

epit

hel,

fibr

obl

met

hod

or

inta

ctL

i et a

l., 2

004

[19]

4 p

IVF

mor

ula

,D

+ F

BS

(bFG

F, L

IF)

inta

ctM

EF

AP

EB

, fib

robl

, neu

ral

blas

tocy

stO

ck e

t al.,

200

5 [3

7]4

pPA

, IV

F, iv

-7d

D +

FB

S (b

FGF,

LIF

)im

mun

osur

gery

STO

AP

Bre

vini

et a

l., 2

005

[38]

PAD

/H

+ K

OSR

+ F

BS

(LIF

)im

mun

osur

gery

STO

Oct

4, N

anog

, *E

B, m

esod

erm

al, e

ndod

erm

al,

neur

oect

oder

mal

Laz

zari

et a

l., 2

005

[48]

no p

ass

IVF

blas

tocy

stD

/H

+KO

SR (L

IF, F

GF)

mic

rosu

rger

y or

inta

ctST

OSh

iue

et a

l., 2

006

[23]

15 p

iv 5

.5–6

.5 d

D +

FB

Sim

mun

osur

gery

STO

AP

EB

inco

rpor

atio

n in

to IC

M o

fth

e ho

st b

last

ocys

tV

acko

va &

Mad

rova

, 200

6 [3

2]19

piv

6–7

dD

+ K

OSR

(LIF

, bFG

F)m

icro

surg

ery

ME

FO

ct4,

Nan

og

Kim

et a

l., 2

007

[39]

5 p

PA, I

VF,

NT

D, D

/H o

r D

/N +

FB

S in

tact

or

imm

unos

urg

ery

STO

, ME

F, P

UC

,A

PE

B, s

pont

aneo

us

dif

fere

ntia

tion

(LIF

)fe

eder

free

Blo

mbe

rg e

t al.,

200

7 [5

4]iv

8 d

D/

M19

9 +

FB

S3

step

dis

sect

ion

STO

Oct

4, N

anog

, **

neu

ro, T

ED

: DM

EM

(hig

h gl

uco

se).

D/

H: D

ME

M (l

ow g

luco

se)/

Ham

’s F

10 m

ediu

m. D

/N

: DM

EM

/N

CSU

-23.

CS:

cal

f ser

um. F

CS:

feta

l cal

f ser

um. F

BS:

feta

l bov

ine

seru

m. K

SOR

: Kno

ckO

ut s

eru

m r

epla

cem

ent.

BR

L: b

uff

alo

rat l

iver

cel

l. C

M:

cond

itio

ned

med

ium

. Iv:

in v

ivo.

NT

: nuc

lear

tran

sfer

. PA

: par

then

ogen

etic

act

ivat

ion.

EB

: em

bryo

id b

ody.

*: +

INF 1

, α-a

myl

ase,

BM

P4

and

neu

rofi

lam

ent.

**: +

SO

X2,

BM

P4,

NO

G, L

IFR

, RE

XO

1, T

DG

F1, D

CX

2 an

d N

CA

M.

VACKOVA et al.1146

animal husbandry.Due to the broad potential use of ES cells, espe-

cially for genetic modifications, there is increasinginterest in establishment of true ES cell lines, notonly from the pig, but also from other livestock. Inthis survey, we attempted to review the currentresults and knowledge concerning ES cell researchin relation to only the pig. Unfortunately, it is stilldifficult to derive and maintain ES cells fromembryos other than those of a few strains of mice[65]. Due to the tremendous amount of research inthis field, we do not mention the results andprogress in other farm animals or the promisingresults concerning use of porcine primordial germcells [74, 80, 81] and somatic stem cells [82, 83, 84].

All three of these types of stem cells can serve as amodel for application of stem cell therapies tohumans, chimera production or nuclear transferapproaches for production of transgenic animalsthat are able to transfer a modified genotype totheir progeny.

Acknowledgments

This work was supported, in part, by MZe0002701401 and MSMT 1M0021620803, CzechRepublic. We thank J. Fulka, Jr. for critical readingof the manuscript.

References

1. Doetschman T, Williams P, Maeda N. Establish-ment of hamster blastocyst-derived embryonic stem(ES) cells. Dev Biol 1988; 127: 224–227.

2. Sukoyan MA, Vatolin SY, Golubitsa AN, Zhele-zova AI, Semanova LA, Serov OL. Embryonic stemcells derived from morulae, inner cell mass and blas-tocysts of mink: Comparisons of their pluripoten-cies. Mol Reprod Dev 1993; 36: 148–158.

3. Graves KH, Moreadith RW. Derivation and charac-terization of putative pluripotential embryonic stemcells from preimplantation rabbit embryo. MolReprod Dev 1993; 36: 424–433.

4. Iannaccone PM, Taborn GU, Garton RL, CapliceMD, Brenin DR. Pluripotent embryonic stem-cellsfrom the rat are capable of producing chimeras. DevBiol 1994; 163: 288–292.

5. Piedrahita JA, Anderson GB, Bondurant RH. Influ-ence of feeder layer type on the efficiency of isola-tion of porcine embryo-derived cell-lines.Theriogenology 1990; 34: 865–877.

6. Piedrahita JA, Anderson GB, Bondurant RH. Onthe isolation of embryonic stem-cells-comparativebehavior of murine, porcine and ovine embryos.Theriogenology 1990; 34: 879–901.

7. Saito S, Strelchenko N, Niemann H. Bovine embry-onic stem cell-like cell-lines cultured over severalpassages. Roux’s Arch Dev Biol 1992; 201: 134–141.

8. Wells DN, Misica PM, Day AM, Tervit HR. Pro-duction of cloned lambs from an established embry-onic cell line: A comparison between in vivo- and invitro-matured cytoplasts. Biol Reprod 1997; 57: 385–393.

9. Meinecke-Tillmann S, Meinecke B. Isolation of ES-like cell lines from ovine and caprine preimplanta-tion embryos. J Anim Breed Genet 1996; 113: 413–426.

10. Saito S, Ugai H, Sawai K, Yamato Y, Minamihashi

A, Kurosaka K, Kobayashi Y, Murata T, Obata Y,Yokoyama K. Isolation of embryonic stem-like cellsfrom equine blastocysts and their differentiation invitro. FEBS Lett 2002; 531: 389–396.

11. Thomson JA, Kalishman J, Golos TG, Durning M,Harris CP, Becker RA, Hearn JP. Isolation of a pri-mate embryonic stem cell line. Proc Natl Acad SciUSA 1995; 92: 7844–7848.

12. Thomson JA, Itskovitz-Eldor J, Shapiro SS,Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM.Embryonic stem cell lines derived from human blas-tocysts. Science 1998; 282: 1145–1147.

13. Prelle K, Holtz W, Sborn M. The intermediate fila-ment protein vimentin as differentiation marker inpreimplantation porcine embryo. Biol Reprod 1995;52 (Suppl 1): 177.

14. Evans MJ, Notarianni E, Laurie S, Moor RM. Deri-vation and preliminary characterization of pluripo-tent cell-lines from porcine and bovine blastocysts.Theriogenology 1990; 33: 125–128.

15. Chung Y, Klimanskaya I, Becker S, Marh J, Lu SJ,Johnson J, Meisner L, Lanza R. Embryonic andextraembryonic stem cell lines derived from singlemouse blastomeres. Nature 2006; 439: 216–219.

16. Mitalipova M, Beyhan Z, First NL. Pluripotency ofbovine embryonic cell line derived from precom-pacting embryos. Cloning 2001; 3: 59–67.

17. Strelchenko N, Verlinsky O, Kukharenko V, Ver-linsky Y. Morula-derived human embryonic stemcells. Reprod Biomed Online 2004; 9: 623–629.

18. Brons IGM, Smithers LE, Trotter MWB, Rugg-Gunn P, Sun BW, Lopes SMCD, Howlett SK,Clarkson A, Ahrlund-Richter L, Pedersen RA, Val-lier L. Derivation of pluripotent epiblast stem cellsfrom mammalian embryos. Nature 2007; 448: 191–195.

1147REVIEW: PORCINE EMBRYONIC STEM CELLS

19. Li M, Li YH, Hou Y, Sun XF, Sun QY, Wang WH.Isolation and culture of pluripotent cells from invitro produced porcine embryos. Zygote 2004; 12: 43–48.

20. Chen LR, Shiue YL, Bertolini L, Medrano JF, Bon-Durant RH, Anderson GB. Establishment of pluri-potent cell lines from porcine preimplantationembryos. Theriogenology 1999; 52: 195–212.

21. Anderson GB, Choi SJ, BonDurant RH. Survival ofporcine inner cell masses in culture and after injec-tion into blastocysts. Theriogenology 1994; 42: 204–212.

22. Hochereau-de-Reviers MT, Perreau C. In-vitro cul-ture of embryonic disc cells from porcine blasto-cysts. Reprod Nutr Dev 1993; 33: 475–483.

23. Shiue YL, Liou JF, Shiau JW, Yang JR, Chen YH,Tailiu JJ, Chen LR. In vitro culture period but notthe passage number influences the capacity of chi-mera production of inner cell mass and its derivingcells from porcine embryos. Anim Reprod Sci 2006;93: 134–143.

24. Ropeter-Scharfenstein M, Neubert N, Prelle K,Holz W. Identification, isolation and culture ofpluripotent cells from the porcine inner cell mass. JAnim Breed Genet 1996; 113: 427–436.

25. Wianny F, Perreau C, deReviers MTH. Prolifera-tion and differentiation of porcine inner cell massand epiblast in vitro. Biol Reprod 1997; 57: 756–764.

26. Strojek RM, Reed MA, Hoover JL, Wagner TE. Amethod for cultivating morphologically undifferen-tiated embryonic stem-cells from porcine blasto-cysts. Theriogenology 1990; 33: 901–913.

27. Gerfen RW, Wheeler MB. Isolation of embryoniccell-lines from porcine blastocysts. Anim Biotech1995; 6: 1–14.

28. Moore K, Piedrahita JA. Effects of heterologoushematopoietic cytokines on in vitro differentiation ofcultured porcine inner cell masses. Mol Reprod Dev1996; 45: 139–144.

29. Moore K, Piedrahita JA. The effects of human leu-kemia inhibitory factor (HLIF) and culture mediumon in vitro differentiation of cultured porcine innercell mass (PICM). In Vitro Cell Dev Biol 1997; 33: 62–71.

30. Li M, Zhang D, Hou Y, Jiao L, Zheng X, Wang WH.Isolation and culture of embryonic stem cells fromporcine blastocysts. Mol Reprod Dev 2003; 65: 429–434.

31. Li M, Ma W, Hou Y, Sun XF, Sun QY, Wang WH.Improved isolation and culture embryonic stemcells from Chinese miniature pig. J Reprod Dev 2004;50: 237–244.

32. Vackova I, Madrova J. Porcine embryonic stem-likecells, an animal model for human stem cell therapy.Hum Reprod 2006; 21 (Suppl 1): 163.

33. Yadav PS, Kues WA, Herrmann D, Carnwath JW,Niemann H. Bovine ICM derived cells express the

Oct4 ortholog. Mol Reprod Dev 2005; 72: 182–190.34. Wang L, Duan EK, Sung LY, Jeong BS, Yang X,

Tian XC. Generation and characterization of pluri-potent stem cells from cloned bovine embryos. BiolReprod 2005; 73: 149–155.

35. Dattena M, Chessa B, Lacerenza D, Accardo C, Pili-chi S, Mara L, Chessa F, Vincenti L, Cappai P. Isola-tion, culture, and characterization of embryonic celllines from vitrified sheep blastocysts. Mol ReprodDev 2006; 73: 31–39.

36. Miyoshi K, Taguchi Y, Sendai Y, Hoshi H, Sato E.Establishment of a porcine cell line from in vitro-pro-duced blastocysts and transfer of the cells into enu-cleated oocytes. Biol Reprod 2000; 62: 1640–1646.

37. Ock SA, Mohana Kumar B, Jin HF, Shi LY, Lee SL,Choe SY, Rho GJ. Establishment of porcine embry-onic stem cell line derived from in vivo blastocysts.Reprod Fertil Dev 2005; 17: 238.

38. Brevini TAL, Cillo F, Gandolfi F. Establishmentand molecular characterization of pig parthenoge-netic embryonic stem cells. Reprod Fertil Dev 2005;17: 235.

39. Kim HS, Son HY, Kim S, Lee GS, Park CH, KangSK, Lee BC, Hwang WS, Lee CK. Isolation and ini-tial culture of porcine inner cell masses derived fromin vitro-produced blastocysts. Zygote 2007; 15: 55–63.

40. Allen ND, Barton SC, Hilton K, Norris ML, SuraniMA. A functional analysis of imprinting in parthe-nogenetic embryonic stem cells. Development 1994;120: 1473–1482.

41. Cibelli JB, Grant KA, Chapman KB, Cunnif K,Worst T, Green HL, Walker SJ, Gutin PH, Vilner L,Tabar V, Dominko T, Kane J, Wettstein PJ, LanzaRP, Studer L, Vrana KE, West MD. Development—parthenogenetic stem cells in nonhuman primates.Science 2002; 295: 819–819.

42. Revazova ES, Turovets NA, Kochetkova OD, Kin-darova LB, Kuzmichev LN, Janus JD, PryzhkovaMV. Patient-specific stem cell lines derived fromhuman parthenogenetic blastocysts. Cloning StemCells 2007; ahead of print.

43. Suemori H, Tada T, Torii R, Hosoi Y, Kobayashi K,Imahie H, Kondo Y, Iritani A, Nakatsuji N. Estab-lishment of embryonic stem cell lines from cynomol-gus monkey blastocysts produced by IVF or ICSI.Dev Dyn 2001; 222: 273–279.

44. Suss-Toby E, Gerecht-Nir S, Amit M, Manor D,Itskovitz-Eldor J. Derivation of a diploid humanembryonic stem cell line from a mononuclearzygote. Hum Reprod 2004; 19: 670–675.

45. Park SP, Lee YJ, Lee KS, Ah Shin H, Cho HY,Chung KS, Kim EY, Lim JH. Establishment ofhuman embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers.Hum Reprod 2004; 19: 676–684.

46. Wakayama S, Jakt ML, Suzuki M, Araki R, HikichiT, Kishigami S, Ohta H, Van Thuan N, Mizutani E,

VACKOVA et al.1148

Sakaide Y, Senda S, Tanaka S, Okada M, MiyakeM, Abe M, Nishikawa SI, Shiota K, Wakayama T.Equivalency of nuclear transfer-derived embryonicstem cells to those derived from fertilized mouseblastocysts. Stem Cells 2006; 24: 2023–2033.

47. Talbot NC, Rexroad CE, Pursel VG, Powel AM,Nel ND. Culturing the epiblast cells of the pig blas-tocyst. In Vitro Cell Dev Biol Anim 1993; 29: 543–554.

48. Lazzari G, Lagutina I, Crotti G, Turini P, ColleoniS, Duchi R, Galli C. Effect of culture system forIVM-IVF pig embryo on the ICMs ability to produceoutgrowths for embryonic stem cell derivation.Reprod Fertil Dev 2005; 17: 237.

49. Solter D, Knowles BB. Immunosurgery of mouseblastocysts. Proc Natl Acad Sci USA 1975; 72: 5099–6005.

50. Whyte A, Bacon M, Ellis S. A monoclonal-antibodyan antigen present on the microvillous membrane ofthe trophectoderm of the preimplantation blastocystof the pig. J Reprod Fert 1984; 71: 599.

51. Prelle K, Wobus AM, Wolf E, Neubert N, Holtz W.Effects of growth factors on the in vitro developmentof porcine inner cell masses isolated by calcium ion-ophore. J Reprod Fert 1994; 13: 41.

52. Talbot NC, Rexroad CE, Pursel VG, Powel AM.Alkaline-phosphatase staining of pig and sheep epi-blast cells in culture. Mol Reprod Dev 1993; 36: 139–147.

53. Talbot NC, Garrett WM. Ultrastructure of theembryonic stem cells of the 8-day pig blastocystbefore and after in vitro manipulation: Developmentof junctional apparatus and the lethal effects of PBSmediated cell-cell dissociation. Anat Rec 2001; 264:101–113.

54. Blomberg LA, Schreier LL, Talbot NC. Expressionanalysis of pluripotency factors in the undifferenti-ated porcine inner cell mass and epiblast during invitro culture. Mol Reprod Dev 2007; ahead of print.

55. Robertson EJ. Embryo derived stem cell lines. In:Robertson EJ (ed.), Teratocarcinomas and Embry-onic Stem Cells: A Practical Approach. Oxford: IRLPress, 1987: 71–122.

56. Prelle K, Wobus AM, Holtz W. Porcine inner cellmass grow undifferentiated in the presence of STOand bFGF. J Reprod Fert 1995; 15: 72.

57. Goldsborough MD, Tilkins ML, Price PJ, Lobo-Alfonso J, Morrison JR, Stevens ME, Meneses J,Pedersen R, Koller B, Latour A. Serum-free cultureof murine embryonic stem (ES) cells. Focus 1998; 20:8–12.

58. Evans MJ, Kaufman MH. Establishment in cultureof pluripotential cells from mouse embryo. Nature1981; 292: 154–156.

59. Strelchenko N. Bovine pluripotent stem cells. Theri-ogenology 1996; 45: 131–140.

60. Mitalipova MM, Rao RR, Hoyer DM, Johnson JA,Meisner LF, Jones KL, Dalton S, Stice SL. Preserv-

ing the genetic integrity of human embryonic stemcells. Nat Biotechnol 2005; 23: 19–20.

61. Wheeler MB. Development and validation of swineembryonic stem-cells: a review. Reprod Fertil Dev1994; 6: 563–568.

62. Notarianni E, Galli C, Laurie S, Moor RM, EvansMJ. Derivation of pluripotent embryonic-cell linesfrom the pig and sheep. J Reprod Fert 1991; 43(Suppl): 255–260.

63. Niwa H, Masui S, Chambers I, Smith AG,Miyazaki J. Phenotypic complementation estab-lishes requirements for specific POU domain andgeneric transactivation function of Oct-3/4 inembryonic stem cells. Mol Cell Biol 2002; 22: 1526–1536.

64. Kirchhof N, Carnwath JW, Lemme E, Anastassia-dis K, Scholer H, Niemann H. Expression pattern ofOct-4 in preimplantation embryos of different spe-cies. Biol Reprod 2000; 63: 1698–1705.

65. Keefer CL, Pant D, Blomberg L, Talbot NC. Chal-lenges and prospects for the establishment ofembryonic stem cell lines of domesticated ungu-lates. Anim Reprod Sci 2007; 98: 147–168.

66. van Eijk MJ, van Rooijen MA, Modina S, Scesi L,Folkers G, van Tol HT, Bevers MM, Fisher SR,Lewin HA, Rakacolli D, Galli C, de Vaureix C,Trounson AO, Mummery CL, Gandolfi F. Molecu-lar cloning, genetic mapping, and developmentalexpression of bovine POU5F1. Biol Reprod 1999; 60:1093–1103.

67. Vejlsted M, Offenberg H, Thorup F, Maddox-Hyt-tel P. Confinement and clearance of OCT4 in theporcine embryo at stereomicroscopically definedstages around gastrulation. Mol Reprod Dev 2006; 73:709–718.

68. Constantinescu D, Gray HL, Sammak PJ, SchattenGP, Csoka AB. Lamin A/C expression is a markerof mouse and human embryonic stem cell differenti-ation. Stem Cells 2006; 24: 177–185.

69. Flechon JF, Delassale S, Guillomot M, ChaudharyN, Galli C, Notarianni E. Expression of nuclearlamins in ovine and porcine blastocysts and embry-onic stem-cells derived from the inner cell mass. JMuscle Res Cell Motil 1991; 12: 493–494.

70. Talbot NC, Worku M, Paape MJ, Grier P, RexroadCE Jr, Pursel VG. Continuous cultures of macroph-ages derived from the 8-day epiblast of the pig. InVitro Cell Dev Biol Anim 1996; 32: 541–549.

71. Kashiwazaki N, Nakao H, Ohtani S, Nakatsuji N.Production of chimeric pigs by the blastocyst injec-tion method. Vet Rec 1992; 130: 186–187.

72. Onishi A. Production of chimeric pigs and the anal-ysis of chimerism using mitochondrial deoxyribo-nucleic-acid as a cell marker. Biol Reprod 1994; 51:1069.

73. Nagashima H, Giannakis C, Ashman RJ, NottleMB. Sex differentiation and germ cell production in

1149REVIEW: PORCINE EMBRYONIC STEM CELLS

chimeric pigs produced by inner cell mass injectioninto blastocysts. Biol Reprod 2004; 70: 702–707.

74. Shim H, Gutierrez-Adan A, Chen LR, BonDurantRH, Behboodi E, Anderson BG. Isolation of pluri-potent stem cells from cultured porcine primordialgerm cells. Biol Reprod 1997; 57: 1089–1095.

75. Golueke PJ, Cibelli, JB, Balise J, Morris J, Kanne JJ,de Leon FAP, Jerry J, Robl JM, Stice SL. Transgenicchimeric pigs derived from cultured embryonic celllines. Theriogenology 1998; 49: 238.

76. Hsu TT, Chen LR, Sheen YW. Comparison of chi-meras production by aggregating demi-embryos inmouse and pigs. J Chin Soc Anim Sci 1990; 19: 27–36.

77. Notarianni E, Laurie S, Ng A, Sathasivan K. Incor-poration of cultured embryonic cells into transgenicand chimeric, porcine fetuses. Int J Dev Biol 1997; 41:537–540.

78. Pain B, Clark ME, Shen M, Nakazawa H, SakuraiM, Samarut J, Etches RJ. Long-term in vitro cultureand characterisation of avian embryonic stem cellswith multiple morphogenetic potentialities. Develop-ment 1996; 122: 2339–2348.

79. Niemann H, Kues W, Carnwath JW. Transgenic

farm animals: present and future. Rev Sci Tech 2005;24: 285–298.

80. Rui R, Qiu Y, Hu YL, Fan B. Establishment of por-cine transgenic embryonic germ cell lines expressingenhanced green fluorescent protein. Theriogenology2006; 65: 713–720.

81. Mueller S, Prelle K, Rieger N, Petznek H, LassingC, Luksh U, Aigner B, Baetscher M, Wolf E, Muel-ler M, Brem G. Chimeric pigs following blastocystinjection of transgenic porcine primordial germcells. Mol Reprod Dev 1999; 54: 244–254.

82. Ringe J, Kaps C, Schmitt B, Büscher K, Bartel J,Smolian H, Schultz O, Burmester GR, Häupl T,Sittinger M. Porcine mesenchymal stem cells -Induction of distinct mesenchymal cell lineages. CellTissue Res 2002; 307: 321–327.

83. Dyce PW, Zhu H, Craig J, Li J. Stem cells with mul-tilineage potential derived from porcine skin. Bio-chem Bioph Res Co 2004; 316: 651–658.

84. Kues WA, Petersen B, Mysegades W, CarnwathJW, Miemann H. Isolation of murine and porcinefetal stem cells from somatic tissue. Biol Reprod 2005;72: 1020–1028.