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Proc Natl Acad Sci U S A. Jul 7, 2009; 106(27): 10993–10998.
Published online Jun 18, 2009. doi:  10.1073/pnas.0905284106
PMCID: PMC2698893
Applied Biological Sciences

Derivation of induced pluripotent stem cells from pig somatic cells

Abstract

For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after ≈22 days, providing an overall reprogramming efficiency of ≈0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of ≈17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.

Keywords: iPS, reprogramming, OCT4

Pluripotent cells from the mouse were first described in 1981 (1). Such cells, which are usually known as embryonic stem cells (ESC), are most commonly derived from either the inner cell mass (ICM) of the blastocyst or from the early epiblast. The cells are truly pluripotent in the sense that they have the potential to differentiate into all of the cell types found in the body and, under appropriate culture conditions, to proliferate more or less indefinitely (2, 3). Their discovery provided a new and powerful model for studying the programs that drive differentiation and senescence. Crucially, murine ESC also afforded a means for “knocking out” and “knocking in” genes in mice by homologous recombination, because mutated cells with the genetic alteration can be introduced into blastocysts to give rise to chimeric mice that, in turn, can pass on the mutated gene through their germline (4, 5). The derivation of human ESC from blastocysts, more than 15 years after the first description of ESC in the mouse (6), presaged their recent derivation from other species, including the dog (7), cat (8), and rat (9, 10). This discovery also heralded the prospects of using ESC in regenerative medicine. Despite their undoubted promise as sources of tissue transplants, many road blocks remain to using human ESC as a source of transplant material, especially as a means to test the efficacy of therapies and the safety of the transferred cells in animals whose anatomy and physiology better resemble the human than the mouse (1115). The pig is a potentially useful model in this regard because of similarities in organ size, immunology, and whole animal physiology (1618).

For reasons that are unclear, the establishment of porcine ESC from ICM of blastocysts and the epiblast of slightly older embryos has proven to be elusive. There has been a similar lack of success with other ungulate species. The earliest reports announcing the derivation of ESC-like cells from ICM of pigs appeared in the early 1990s (1921), but these ESC-like cells and many others since then, including ones for cattle, goat, and sheep, as well as for pig, have failed to meet the full criteria to define them as ESC (1113). There are a number of reasons that might explain the problems encountered, including choice of the wrong stage of embryo development to establish the cultures, inappropriate culture and cell passage conditions, and contamination by more vigorously growing cells, such as the endoderm and trophectoderm of the blastocyst from which the culture was derived. Attempts to create pluripotent cells from embryonic germ cells have also run into difficulties. As a result, this field of research has languished without major breakthroughs for well over a decade. These difficulties, as well as the potential value of ESC from ungulate species, have been discussed at length in several recent reviews (11, 1315, 22).

Many recent papers (2329) have reported the generation of induced pluripotent stem cells (iPSC), with properties almost indistinguishable from those of ESC, by transgenic manipulation of mouse and human somatic cells. In most of these examples, the same 4 “reprogramming genes” were able to establish the iPSC (25, 26). In addition, the efficiency of the overall process could be improved by the inclusion of various small molecules and growth factors (30). The major focus of this report describes the derivation of porcine-induced pluripotent stem cells (piPSC) based on the same strategy as that used for the mouse and human, namely ectopic expression of reprogramming genes in somatic cells by the use of lentiviral vectors. An important justification for establishing such a technology is that the ability to derive iPSC from a particular pig, conduct tissue transplantation on the same animal at a later time, and then follow the success of the transplant over the course of months or even years would be a particularly valuable advance. Finally, the ability to provide iPSC from animals with valuable traits would provide a permanent source of cells for clonal propagation that would likely avoid the inefficiencies and problems arising from somatic cell nuclear transfer (SCNT), where many of the cloned offspring die or are developmentally abnormal even if they survive to term (31).

Results

Generation of piPSC.

Four reprogramming genes (OCT4, SOX2, KLF4, and c-MYC), each integrated into separate lentiviral vectors, were introduced into porcine fetal fibroblasts (PFF) and PFF expressing enhanced green fluorescent protein (EGFP) (Fig. 1A and Fig. S1). Twenty-two days after transduction, compact colonies comprised of cells, which were positive for alkaline phosphatase (AP), stage-specific embryonic antigen (SSEA)1, and OCT4, were noted (Fig. 1 B–F). Individual colonies were mechanically dissociated by using a pulled Pasteur pipette. A colony's component cells were transferred to fresh medium in 24-well plates coated with irradiated MEF. After ≈4 days, well-developed secondary colonies (Fig. 1C), resembling hESC colonies, formed. The cells exhibited a high nuclear to cytoplasm ratio with prominently visible nucleoli (Fig. 1D). This protocol was used to isolate a large number of clonal lines (65 under 20% O2 conditions, 96 under 4% O2), each derived from a single ESC-like colony. Similar to hESC, areas of large, flattened cells, presumably undergoing spontaneous differentiation, became evident within some colonies, particularly if the cells were not passaged within 5 days (Fig. 1G).

Fig. 1.
piPSC colonies derived from PFF. (A) A phase contrast image of PFF. (B) Phase contrast image of granulated piPSC similar to mouse and human iPSC begin to emerge ≈3 weeks after viral infection. (C) A representative piPSC colony after serial passage ...

All of the cell lines grew at similar rates, requiring subculture at a roughly 1:10 ratio every 4–5 days. The ID6 line, the only one to be carefully examined, showed a doubling time of ≈17 h.

Expression of Pluripotent Genes in piPSC.

Several genes associated with pluripotency and known to be expressed in the ICM of pig embryos, e.g., OCT4, SOX2, TDGF1 (CRIPTO) and TERT (32, 33), were expressed only in the piPSC, and not in the founder PFF (Fig. 2), providing evidence that reprogramming into a pluripotent state had been successful. Results with NANOG were more equivocal, because expression was also observed in the PFF. Nevertheless, as indicated by semiquantitative RT-PCR, NANOG was up-regulated in the putative piPSC relative to the PFF (Fig. 2), again consistent with reprogramming. c-MYC and KLF4 expression was detected in the 3 piPSC lines examined, but again both genes were also prominently expressed in the PFF (Fig. 2A). Assessments of c-MYC and KLF4 expression were complicated by the fact that the primers designed to amplify the porcine transcripts were also able to detect human c-MYC and KLF4 transcripts in hESC (Fig. 2A) and potentially in the piPSC, if the reprogramming genes continued to be expressed. To test this possibility and to distinguish between the pig and human transcripts, we designed forward primers specific for the human genes and a common reverse primer corresponding to the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) region of the lentiviral vector (Table S1). PCR of reverse-transcribed piPSC RNA with these primers clearly showed that all 4 human transgenes continued to be coexpressed along with their porcine orthologs in reprogrammed piPSC (Fig. S2A).

Fig. 2.
Gene expression analysis of piPSC. (A) RT-PCR analysis for expression of selected pluripotency genes in piPSC, PFF, and H9 hESC. The primers were chosen for their specificity toward the porcine (p) genes rather than their human orthologs, but those for ...

Microarray Analysis.

Transcript profiling comparisons that used Affymetrix porcine microarrays (GEO accession GSE15472) were conducted to provide further confirmation that reprogramming of PFF had occurred and to determine the degree of similarity between different piPSC lines. Hierarchical clustering of all of the 8,015 genes that were either up- or down-regulated with a P value of ≤0.05 showed that the 3 piPSC lines clustered together in terms of their overall gene expression and were well-separated from PFF lines (Fig. 2B), although one of the piPSC lines, ID6, appeared as a relative outlier compared with the other two. Of these genes, 2,605 (out of a total of 20,201 represented on the array) displayed at least a 2-fold and 4,297 at least a 1.3-fold increase in expression (P ≤ 0.05), whereas over 3,700 genes displayed significantly decreased expression in the piPSC relative to the PFF.

Several genes associated with the pluripotent state in mouse and human pluripotent cells, including SOX2 and NANOG, were either not represented on the pig array or not annotated. However, the regulation of many other genes associated with pluripotency could be assessed from the microarray data. The log2 fold changes in expression values for these genes are shown in Fig. 2C. As anticipated, the majority of such genes were up-regulated in the piPSC relative to the PFF. These genes included CDH1, PODXL, LIN28, GCNF, TNAP, GNL3, CD9, ZFP42, UTF1, and, as demonstrated in the previous section, OCT4. However, c-MYC and KLF4 were down-regulated in piPSC relative to PFF (Fig. 2C; ≈97% and 75%, respectively), data that are consistent with the RT-PCR analyses (Fig. 2A). In contrast to c-MYC, mRNA for MYCN was barely detectable in PFF, but displayed a 102-fold up-regulation in piPSC.

Immunocytochemistry.

piPSC display many of the same protein and carbohydrate antigens as mouse and human ESC. As anticipated from RT-PCR and microarray data, antibodies against OCT4, NANOG, and SOX2 reacted positively with the cells of piPSC colonies (Fig. 3 and SI Materials and Methods) and negatively with the progenitor PFF cells (Fig. S3B). The cells were also positive for the cell surface marker SSEA-1, but displayed very weak to negative staining for SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81 (Fig. S3).

Fig. 3.
Immunofluorescence staining of pluripotent markers. The immunofluorescence staining of pluripotent markers OCT4 (A Upper), NANOG (B Upper), and SOX2 (C Upper) in piPSC colonies cultured on MEF are shown. (A–C Lower) Specific localization to nuclei ...

Telomerase Activity of piPSC.

All of the 5 piPSC lines tested so far expressed comparable amounts of telomerase (TERT) activity (Fig. 4), a hallmark of mESC (34). The activity in the piPSC was at least 5-fold higher than in the hESC line (H9), whereas expression was barely detectable above background in the founder PFF and the feeder MEF cells (Fig. 4).

Fig. 4.
Telomerase activity in piPSC. Telomerase activities in piPSC lines (IC1 passage10, ID4 p10, ID6 p10, IIIB2 p3, IB3 p8) are compared with their parental EGFP-PFF p10, MEF p4, and H9 hESC p41. The assay was performed in triplicate samples with 0.2 μg ...

Embryoid Body (EB) Formation and Real-Time RT-PCR Analysis.

We tested whether piPSC could form EB on culture in the absence of FGF2 on a nonadhesive substratum. Nine piPSC lines were examined, and all, including the three analyzed in Fig. 2, differentiated into EB under such conditions. When these EB were subsequently placed on a gelatin coated surface and cultured in presence of either BMP4, FBS, or RA for 4 additional days, they attached to the substratum, began to spread, and displayed overt signs of differentiation. The appearance of the IIIB2 line exposed to 5% FBS is shown in Fig. 5A.

Fig. 5.
Differentiation of piPSC into embryoid bodies (EB). (A) Day 0 shows piPSC plated on MEF. Day 1 represents an image of the derived EB the next day and Day 5 represents an image after 5 days under differentiation conditions. Day 9 shows cells treated with ...

Real-time RT-PCR analysis of RNA isolated from piPSC lines before their conversion to EB and following exposure of the resulting EB to BMP4, FBS, and RA revealed that OCT4 was almost completely silenced and SOX2 strongly down-regulated by all of the treatments. By contrast, candidate genes normally characteristic of trophectoderm (CDX2 in ID6 and PTI in ID4 and ID6) and the 3 germ layers (endoderm, AFP in all three, NCSTN in IC1 and ID6; mesoderm, DES in ID6, ectoderm, CRABP2 in all three) were up-regulated (Fig. 5B).

Karyotype Analysis.

One of the potential roadblocks in using iPSC for therapeutics is acquisition of chromosomal abnormalities. Of the 2 piPSC lines so far subjected to cytogenetic analysis, one (ID6) exhibited a normal karyotype in 19 of 20 cells at passage 18, whereas the other (IIIC6) had a paracentric inversion in chromosome 16 in 19 of 20 metaphase spreads (Fig. S4), a change that was not evident in the original PFF (Fig. S4).

Teratoma Formation.

To test for pluripotency in vivo, we transplanted piPSC (clone ID6) s.c. into dorsal flanks of immunodeficient (CD-1 nude) mice (n = 2). Three months after injection, one mouse had developed a small, solid tumor ≈2 × 3 mm in size that on histological examination was found to be minimally differentiated. The second mouse had developed several larger, solid tumors lodged within its peritoneal cavity, possibly because of poor placement of the injection needle. These tumors contained various kinds of tissue (Fig. 6), including neural epithelium (ectoderm), striated muscle (mesoderm), and epithelium with brush border (endoderm). The tumors also included neural fiber (ectoderm), connective tissue, and adipose-like tissue (mesoderm) (Fig. S5). RT-PCR confirmed that these teratomas were of porcine and not murine origin (Fig. S5). Nude mice (n = 2) injected with a comparable number of PFF cells did not develop tumors.

Fig. 6.
Histological section of a solid, encapsulated tumor removed from the peritoneum of a nude mouse that had been injected s.c. with cells from piPSC line ID6. The highly differentiated tumor contained a wide range of tissues, including neural epithelium ...

Discussion

Since the initial generation of murine iPSC (26), there have been numerous follow-up papers describing iPSC from both mouse and human cells. These studies have used both embryonic and adult cells and different combinations of reprogramming genes (23, 2526, 2930, 35, 3741). More recently, iPSC have been generated from monkey (42), rat (43), and, while this manuscript was under review, miniature swine (44). This technology might be especially valuable in agriculturally important species for nuclear transfer experiments, as pluripotent cells might provide higher cloning efficiency and avoid the abnormalities and deaths associated with using differentiated somatic cells (45). In addition, it might be possible to establish gene targeting technologies with the ultimate goal of creating genetic models for human diseases in species where mouse models are inappropriate. Here, we have derived iPSC from the pig with the same OSKM genes described in an original report for mouse fibroblasts (26). Reprogramming efficiency appeared to be quite high, as we were able to isolate ≈100 iPSC colonies from 105 transduced PFF. Whether other gene combinations will prove to be as efficient as OSKM, and whether there will be the same ease of reprogramming with other somatic cell types, remains to be seen.

Initially, we investigated whether the presumed piPSC shared characteristics of pluripotent stem cells from other sources. As with human and mouse, a feeder layer of irradiated MEF was essential for maintenance of stem cell properties, but, unlike the mouse, this requirement could not be substituted by the addition of LIF to the medium. The lack of LIF dependency is underscored by the down-regulation of LIF receptor gene, LIFR, during reprogramming (GEO accession GSE15472). As with human cells, factors produced by MEF in combination with FGF2 may be needed to maintain pluripotency and prevent differentiation. On the other hand, the display of surface marker SSEA-1 and the absence of SSEA-3 and -4, are features of murine iPSC and ESC and not of human pluripotent cells. The piPSC also lack TRA-1-60 and TRA-1-81, which are characteristic of human cells (46, 47). The expression of SSEA-1 is consistent with reports showing this antigen to be expressed on cells of ICM of d 7 pig blastocysts (13, 22, 32, 48) and primordial germ cells of d 18 to 26 conceptuses, which also lack SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (48, 49). Together, these data are consistent with the pluripotent nature of piPSC and their resemblance to ESC. Another feature common to piPSC and human and murine ESC is their relatively rapid rate of proliferation. The calculated 17-h linear-phase doubling time is slightly greater than that of mouse (50) ESC and roughly similar to that of human (51).

Pluripotent stem cells can extend their growth through many doublings without signs of senescence (52). Although the majority of our cell lines have not been taken beyond a few passages, line ID6, which demonstrates a consistent stem cell-like phenotype, a normal karyotype, and telomerase activity higher than the human ESC line, H9 (Fig. 4 and Fig. S4), has been subcultured 40 times at a ≈1:10 passage ratio at roughly 5-day intervals, which represents at least ≈224 doublings. As with hESC, all of the piPSC lines, including ID6, have a tendency to differentiate spontaneously, especially if they are maintained without passage for more than a week or cultured at high cell density.

The 3 piPSC expressed much the same spectrum of pluripotent genes, including OCT4, SOX2, CDH1, LIN28, UTF1, and ZFP42, as reported for human and murine ESC and iPSC (53). Microarray analysis of the human ESC lines H1 from this laboratory, grown under identical conditions to the piPSC, confirms the similarity of the hESC to iPSC in terms of overall patterns of gene expression, despite the complications arising from the incomplete annotation of the porcine array (GEO accession GSE9510, 10469, and 15472). In addition, there was up-regulation of expression of other genes, which, although not categorized as specific to pluripotent cells, are associated with reprogramming of human and murine iPSC (Fig. S6) (54). Further evidence that the PFF had been efficiently reprogrammed is the very low expression of the differentiation-associated, fibroblast-specific gene, THY1, in piPSC, which is rapidly down-regulated when murine fibroblasts are reprogrammed (54). To our surprise, porcine c-MYC and KLF4 were down-regulated, whereas MYCN was strongly up-regulated in piPSC compared with PFF (Fig. 2B), but examination of the microarray data of others (23, 54) revealed that a similar phenomenon has been observed in reprogramming of murine and human cells. Finally, pNANOG was up-regulated during reprogramming, although it was already expressed in the parental PFF cells, an observation consistent with previous reports that NANOG is not a reliable marker for pluripotency in pig (32).

Until recently most iPSC have been generated by using integrating retroviral vectors, leading in some cases, at least, to the continued expression of the transgenes in the reprogrammed cells (29, 5559). As the ectopically expressed reprogramming genes may cause imbalance in the pluripotency network and even influence the ability of reprogrammed cells to differentiate optimally (29, 54, 59), ideally the genes should either not be permitted to integrate in the first place (6062) or excised once reprogramming has been completed (59, 63). A more recent strategy has been to provide the reprogramming proteins themselves directly to the targeted cell type (64) Because retroviruses were used in our experiments, not unexpectedly, transcripts for the 4 transgenes remained detectable in the piPSC (Fig. S2), just as they did in the iPSC established from PFF of miniature swine (44). Although such expression is not desirable, an impaired ability to differentiate was not a feature of iPSC described here, which were fully capable of generating EB expressing genes indicative of the 3 germ layers and trophoblast (Fig. 5B) and of producing highly differentiated teratomas in immune-compromised mice (Fig. 6 and Fig. S5). Nevertheless, if these cells are to be tested in pigs to mimic therapeutic applications in human patients, it may be necessary to ensure that transgene expression is eliminated completely from the donor lines used by using one or other of the strategies described above.

Materials and Methods

Cell Culture and Lentiviral Transduction of Human Factors.

The ORFs of the human (h) SOX2, hKLF4, and hc-MYC were cloned into the FUGW lentiviral vector (65), and the hOCT4 cDNA was cloned into pSIN18 cPPt.hEF1a.EGFP.WPRE (66) (Fig. S1). Pseudovirus was produced in human 293FT cells (Invitrogen) by transfection with each lentiviral vector along with the VSV-G envelope (pMD2.G) and packaging vector (psPAX2) (67) by using Lipofectamine and PLUS reagents (Invitrogen). Titered virus (68) was used to infect the target cells (PFF or EGFP-PFF; 1 × 105 cells/35-mm dish). On day 2 after infection, the cells were dispersed with trypsin and transferred to 10-cm plates seeded with irradiated MEF. Subsequently, cells were maintained on a culture medium standardized for human ESC (69) containing 4 ng/ml human FGF2 (Peprotech).

Alkaline Phosphatase (AP) Staining and Immunocytochemistry (ICC).

AP staining was performed with the AP detection kit (Chemicon). For immunocytochemistry, cells were fixed in 2% paraformaldehyde in PBS for 20 min at room temperature (RT), washed, and exposed to either 5% goat or donkey serum (Sigma), 1% BSA (Jackson-Immunoresearch), and 0.1% Triton X-100 (Fisher) in PBS for 30 min. The cells were then incubated with primary antibody (SI Materials and Methods) overnight at 4 °C. After washing, the cells were incubated with secondary antibody (SI Materials and Methods). Nuclei were stained with 6 ng/ml DAPI (Invitrogen).

Transcriptional Profiling by Microarray.

RNA from 3 piPSC and 2 PFF lines was extracted by using STAT-60 (IsoTex Diagnostics). Ten-μg of each sample was subjected to double-stranded cDNA synthesis and labeling by using the GeneChip Expression 3′-Amplification IVT Labeling Kit (Affymetrix), according to manufacturer's instructions. Biotin-labeled cRNA was fragmented and hybridized to the Affymetrix GeneChip Porcine Genome Array. Microarrays were scanned (GeneChip Scanner 3000; Affymetrix), and data were assembled by MicroArray Suite 5.0 software (Affymetrix). The output data were analyzed with GeneSpring GX analysis software (Agilent). The CEL files for each array were normalized, and false discovery rates were calculated by T test unpaired, unequal variance with asymptotic p-values and Benjamini-Hochberg multiple testing correction options. Fold-changes in pairwise comparison of the piPSC and PFF data were calculated based on the unadjusted data means to obtain a list of 8,015 genes with an acceptable fold-change of 1.3. Clustering was performed by the Pearson-centered similarity measure and single linkage rule. Because the porcine array remains poorly annotated, additional annotation was performed by using the information of Tsai et al. (70) (Gene Expression Omnibus accession no. GPL6472) and an annotated file provided by Dr. C. K. Tuggle (Iowa State University, Ames, IA) (36).

Embryoid Body (EB) Formation.

Colonies were detached from the MEF feeder layer by manual dissection and transferred into hESC medium without FGF2 (differentiation medium) in low attachment plates (Corning). After 5 days of culture, the EB were transferred onto adherent, gelatin-coated tissue-culture dishes and were cultured in differentiation medium supplemented with either 10 ng/ml BMP4 (R & D Systems), 5% FBS, or 0.5 μM all-trans retinoic acid (RA; Sigma) and harvested for RNA isolation 9 days later.

Real-Time RT-PCR Analysis.

Real-time RT-PCR was performed on RNA extracted from EB (at day 9), undifferentiated piPSC, and PFF to assess the relative concentrations of genes from the 3 germ layers [AFP and NCSTN (endoderm), CRABP2 (ectoderm), and DES (mesoderm)] as well as pluripotent (OCT4 and SOX2), and trophoblast genes (CDX2 and PTI). A reference sample constituted by pooling RNA from all of the samples was also used in the analysis. Three μg of DNase-treated RNA was used in the first strand cDNA synthesis (qScript cDNA synthesis kit; Quanta Biosciences). Validated primer sets for real-time PCR analysis are listed in Table S1. Expression of candidate genes relative to GAPDH in the test was determined with SYBR Green PCR Master Mix (Applied Biosystems) on an ABI PRISM 7500 Real Time System (Applied Biosystems). The conditions for real-time RT-PCR were as follows: 50 °C, 2 min; 95 °C,10 min; followed by 40 amplification cycles (95 °C,15 s; 60 °C (62 °C for OCT4), 30 s). The reaction was terminated by an elongation and data acquisition step at 75 °C for 1 min. The relative expression ratio of target genes was calculated by ΔΔCt method. The specificity of the PCR products amplified was confirmed by dissociation curve analysis.

Telomerase Activity Assay.

Telomerase activity in 0.2-μg cell protein from representative piPSC, MEFs, and HESC was determined by using TRAPEZE RT Telomerase Detection Kit (Chemicon) with Platinum TaqDNA polymerase (Invitrogen) according to manufacturer's instructions.

Teratoma Formation.

106 ID6 cells from a confluent 35-mm dish were injected s.c. into dorsal flanks of two 4-week-old CD-1 nude mice, Crl: CD-1-Foxn1nu (Charles River). Tumors were harvested 3 months after injection, dissected, and fixed in 4% paraformaldehyde. Paraffin-embedded tissue was sectioned and stained with hematoxylin and eosin. All animal experiments were approved by the Institutional Animal Care and Use Committee under Protocol No. 4467. Control animals were injected with an equivalent number of PFF cells.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Dr. Randall Prather and Lee Spate for providing EGFP-PFF and PFF, Drs. Michal Gropp and Benjamin Reubinoff (Hadassah University Medical Center, Jerusalem, Israel) for the pSIN18.cPPT, hEF1a.EGFP.WPRE plasmid, Dr. David Baltimore (California Institute of Technology, Pasadena, CA) for the FUGW and VSVG envelope plasmids, Dr. Didier Trono (Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland) for the psPAX2 packaging plasmid, and Dr. Christopher K. Tuggle (Iowa State University, Ames, IA) for information on annotation of porcine EST sequences. Kristin Whitworth assisted in the microarray analysis and Dr. Kei Kuroki (Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, Missouri) provided histological analysis of the teratomas. This work was supported by National Institutes of Health Grants HD42201 and HD21896 (to R.M.R), and Missouri Life Sciences Trust Fund Grant 00022147 (to T.E.).

Footnotes

The authors declare no conflict of interest.

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE15472).

This article contains supporting information online at www.pnas.org/cgi/content/full/0905284106/DCSupplemental.

References

1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154–156. [PubMed]
2. Evans M. Embryonic stem cells: A perspective. Novartis Found Symp. 265:98–103. and discussion (2005) 265:103–106, 265:122–128. [PubMed]
3. Wobus AM, Boheler KR. Embryonic stem cells: Prospects for developmental biology and cell therapy. Physiol Rev. 2005;85:635–678. [PubMed]
4. Thomas KR, Folger KR, Capecchi MR. High frequency targeting of genes to specific sites in the mammalian genome. Cell. 1986;44:419–428. [PubMed]
5. Doetschman T, et al. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987;330:576–578. [PubMed]
6. Thomson JA, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147. [PubMed]
7. Hatoya S, et al. Isolation and characterization of embryonic stem-like cells from canine blastocysts. Mol Reprod Dev. 2006;73:298–305. [PubMed]
8. Yu X, et al. Isolation and characterization of embryonic stem-like cells derived from in vivo-produced cat blastocysts. Mol Reprod Dev. 2008;75:1426–1432. [PubMed]
9. Buehr M, et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell. 2008;135:1287–1298. [PubMed]
10. Li P, et al. Germline competent embryonic stem cells derived from rat blastocysts. Cell. 2008;135:1299–1310. [PMC free article] [PubMed]
11. Vackova I, Ungrova A, Lopes F. Putative embryonic stem cell lines from pig embryos. J Reprod Dev. 2007;53:1137–1149. [PubMed]
12. Keefer CL, Pant D, Blomberg L, Talbot NC. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim Reprod Sci. 2007;98:147–168. [PubMed]
13. Brevini TA, et al. Porcine embryonic stem cells: Facts, challenges and hopes. Theriogenology. 2007;68(Suppl 1):S206–S213. [PubMed]
14. Talbot NC, Blomberg le A. The pursuit of ES cell lines of domesticated ungulates. Stem Cell Rev. 2008;4:235–254. [PubMed]
15. Hall V. Porcine embryonic stem cells: A possible source for cell replacement therapy. Stem Cell Rev. 2008;4:275–282. [PubMed]
16. Prather RS, et al. Transgenic swine for biomedicine and agriculture. Theriogenology. 2003;59:115–123. [PubMed]
17. Brandl U, et al. Transgenic animals in experimental xenotransplantation models: Orthotopic heart transplantation in the pig-to-baboon model. Transplant Proc. 2007;39:577–578. [PubMed]
18. Piedrahita JA, Mir B. Cloning and transgenesis in mammals: Implications for xenotransplantation. Am J Transplant. 2004;4(Suppl 6):43–50. [PubMed]
19. Notarianni E, Laurie S, Moor RM, Evans MJ. Maintenance and differentiation in culture of pluripotential embryonic cell lines from pig blastocysts. J Reprod Fertil Suppl. 1990;41:51–56. [PubMed]
20. Piedrahita JA, Anderson GB, Bondurant RH. On the isolation of embryonic stem cells: Comparative behavior of murine, porcine, and ovine embryos. Theriogenology. 1990;34:879–901. [PubMed]
21. Strojek RM, Reed MA, Hoover JL, Wagner TE. A method for cultivating morphologically undifferentiated embryonic stem cells from porcine blastocysts. Theriogenology. 1990;33:901–913. [PubMed]
22. Brevini TA, et al. Derivation and characterization of pluripotent cell lines from pig embryos of different origins. Theriogenology. 2007;67:54–63. [PubMed]
23. Lowry WE, et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA. 2008;105:2883–2888. [PMC free article] [PubMed]
24. Nakagawa M, et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotech. 2008;26:101–106. [PubMed]
25. Takahashi K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872. [PubMed]
26. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. [PubMed]
27. Park I-H, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 2008;451:141–146. [PubMed]
28. Kim JB, et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature. 2008;454:646–650. [PubMed]
29. Yu J, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318:1917–1920. [PubMed]
30. Amabile G, Meissner A. Induced pluripotent stem cells: Current progress and potential for regenerative medicine. Trends Mol Med. 2009;15:59–68. [PubMed]
31. Prather RS, Sutovsky P, Green JA. Nuclear remodeling and reprogramming in transgenic pig production. Exp Biol Med. 2004;229:1120–1126. [PubMed]
32. Blomberg L, Schreier LL, Talbot NC. Expression analysis of pluripotency factors in the undifferentiated porcine inner cell mass and epiblast during in vitro culture. Mol Reprod Dev. 2008;75:450–463. [PubMed]
33. Magnani L, Cabot RA. In vitro and in vivo derived porcine embryos possess similar, but not identical, patterns of Oct4, Nanog, and Sox2 mRNA expression during cleavage development. Mol Reprod Dev. 2008;75:1726–1735. [PubMed]
34. Armstrong L, et al. mTert expression correlates with telomerase activity during the differentiation of murine embryonic stem cells. Mech Dev. 2000;97:109–116. [PubMed]
35. Aoi T, et al. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science. 2008;321:699–702. [PubMed]
36. Wang Y, et al. Analysis of porcine transcriptional response to Salmonella enterica serovar Choleraesuis suggests novel targets of NFkappaB are activated in the mesenteric lymph node. BMC Genomics. 2008;9:437. [PMC free article] [PubMed]
37. Park IH, et al. Generation of human-induced pluripotent stem cells. Nat Protoc. 2008;3:1180–1186. [PubMed]
38. Maherali N, et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell. 2007;1:55–70. [PubMed]
39. Wernig M, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007;448:318–324. [PubMed]
40. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007;448:313–317. [PubMed]
41. Feng B, et al. Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nat Cell Biol. 2009;11:197–203. [PubMed]
42. Liu H, et al. Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell. 2008;3:587–590. [PubMed]
43. Liao J, et al. Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell. 2009;4:11–15. [PubMed]
44. Esteban MA, et al. Generation of induced pluripotent stem cell lines from tibetan miniature pig. J Biol Chem. 2009 Apr 17; doi: 10.1074/jbc.M109.008938. [PMC free article] [PubMed] [Cross Ref]
45. Oback B, Wells DN. Donor cell differentiation, reprogramming, and cloning efficiency: Elusive or illusive correlation? Mol Reprod Dev. 2007;74:646–654. [PubMed]
46. Draper JS, Pigott C, Thomson JA, Andrews PW. Surface antigens of human embryonic stem cells: Changes upon differentiation in culture. J Anat. 2002;200:249–258. [PMC free article] [PubMed]
47. O'Connor MD, et al. Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem Cells. 2008;26:1109–1116. [PubMed]
48. Wianny F, Perreau C, Hochereau de Reviers MT. Proliferation and differentiation of porcine inner cell mass and epiblast in vitro. Biol Reprod. 1997;57:756–764. [PubMed]
49. Takagi Y, Talbot NC, Rexroad CE, Jr, Pursel VG. Identification of pig primordial germ cells by immunocytochemistry and lectin binding. Mol Reprod Dev. 1997;46:567–580. [PubMed]
50. Udy GB, Parkes BD, Wells DN. ES cell cycle rates affect gene targeting frequencies. Exp Cell Res. 1997;231:296–301. [PubMed]
51. Becker KA, et al. Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol. 2006;209:883–893. [PubMed]
52. Zeng X. Human embryonic stem cells: Mechanisms to escape replicative senescence? Stem Cell Rev. 2007;3:270–279. [PubMed]
53. Ginis I, et al. Differences between human and mouse embryonic stem cells. Dev Biol. 2004;269:360–380. [PubMed]
54. Sridharan R, et al. Role of the murine reprogramming factors in the induction of pluripotency. Cell. 2009;136:364–377. [PMC free article] [PubMed]
55. Dimos JT, et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 2008;321:1218–1221. [PubMed]
56. Ebert AD, et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature. 2009;457:277–280. [PMC free article] [PubMed]
57. Hockemeyer D, et al. A drug-inducible system for direct reprogramming of human somatic cells to pluripotency. Cell Stem Cell. 2008;3:346–353. [PMC free article] [PubMed]
58. Park IH, et al. Disease-specific induced pluripotent stem cells. Cell. 2008;134:877–886. [PMC free article] [PubMed]
59. Soldner F, et al. Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell. 2009;136:964–977. [PMC free article] [PubMed]
60. Okita K, et al. Generation of mouse induced pluripotent stem cells without viral vectors. Science. 2008;322:949–953. [PubMed]
61. Stadtfeld M, et al. Induced pluripotent stem cells generated without viral integration. Science. 2008;322:945–949. [PMC free article] [PubMed]
62. Yu J, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009;324:797–801. [PMC free article] [PubMed]
63. Yusa K, Rad R, Takeda J, Bradley A. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods. 2009;6:363–369. [PMC free article] [PubMed]
64. Zhou H, et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell. 2009;4(5):381–384. [PubMed]
65. Lois C, et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 2002;295:868–872. [PubMed]
66. Gropp M, et al. Stable genetic modification of human embryonic stem cells by lentiviral vectors. Mol Ther. 2003;7:281–287. [PubMed]
67. Szulc J, et al. A versatile tool for conditional gene expression and knockdown. Nat Methods. 2006;3:109–116. [PubMed]
68. Lizee G, et al. Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression. Hum Gene Ther. 2003;14:497–507. [PubMed]
69. Amit M, et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol. 2000;227:271. [PubMed]
70. Tsai S, et al. Annotation of the Affymetrix porcine genome microarray. Anim Genet. 2006;37:423–424. [PubMed]

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