Derivation of Induced Pluripotent Stem Cells from Pig Somatic Cells

Derivation of induced pluripotent stem cells from pig somatic cells

Toshihiko Ezashia, Bhanu Prakash V. L. Telugua, Andrei P. Alexenkoa, Shrikesh Sachdevb, Sunilima Sinhaa, and R. Michael Robertsa,b,1

Divisions of aAnimal Sciences and bBiochemistry, University of Missouri, Columbia, MO 65211

Contributed by R. Michael Roberts, May 13, 2009 (sent for review April 15, 2009) For reasons that are unclear the production of embryonic stem cells For reasons that are unclear, the establishment of porcine ESC from ungulates has proved elusive. Here, we describe induced from ICM of blastocysts and the epiblast of slightly older pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts embryos has proven to be elusive. There has been a similar lack by lentiviral transduction of 4 human (h) genes, hOCT4,hSOX2, of success with other ungulate species. The earliest reports hKLF4, and hc-MYC, the combination commonly used to create iPSC announcing the derivation of ESC-like cells from ICM of pigs in mouse and human. Cells were cultured on irradiated mouse appeared in the early 1990s (19–21), but these ESC-like cells and embryonic fibroblasts (MEF) and in medium supplemented with many others since then, including ones for cattle, goat, and knockout serum replacement and FGF2. Compact colonies of alka- sheep, as well as for pig, have failed to meet the full criteria to line phosphatase-positive cells emerged after Ϸ22 days, providing define them as ESC (11–13). There are a number of reasons that an overall reprogramming efficiency of Ϸ0.1%. The cells expressed might explain the problems encountered, including choice of the porcine OCT4, NANOG, and SOX2 and had high telomerase activity, wrong stage of embryo development to establish the cultures, inappropriate culture and cell passage conditions, and contam- but also continued to express the 4 human transgenes. Unlike ination by more vigorously growing cells, such as the endoderm human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but and trophectoderm of the blastocyst from which the culture was negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix derived. Attempts to create pluripotent cells from embryonic (porcine) microarrays and real time RT-PCR supported the conclu- germ cells have also run into difficulties. As a result, this field of SCIENCES sion that reprogramming to pluripotency was complete. One cell research has languished without major breakthroughs for well line, ID6, had a normal karyotype, a cell doubling time of Ϸ17 h, over a decade. These difficulties, as well as the potential value of APPLIED BIOLOGICAL and has been maintained through >220 doublings. The ID6 line ESC from ungulate species, have been discussed at length in formed embryoid bodies, expressing genes representing all 3 germ several recent reviews (11, 13–15, 22). layers when cultured under differentiating conditions, and tera- Many recent papers (23–29) have reported the generation of tomas containing tissues of ectoderm, mesoderm, and endoderm induced pluripotent stem cells (iPSC), with properties almost origin in nude mice. We conclude that porcine somatic cells can be indistinguishable from those of ESC, by transgenic manipulation of reprogrammed to form piPSC. Such cell lines derived from individ- mouse and human somatic cells. In most of these examples, the ual animals could provide a means for testing the safety and same 4 ‘‘reprogramming genes’’ were able to establish the iPSC (25, efficacy of stem cell-derived tissue grafts when returned to the 26). In addition, the efficiency of the overall process could be same pigs at a later age. improved by the inclusion of various small molecules and growth factors (30). The major focus of this report describes the derivation iPS ͉ reprogramming ͉ OCT4 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 luripotent cells from the mouse were first described in 1981 (1). use of lentiviral vectors. An important justification for establishing PSuch cells, which are usually known as embryonic stem cells such a technology is that the ability to derive iPSC from a particular (ESC), are most commonly derived from either the inner cell mass pig, conduct tissue transplantation on the same animal at a later (ICM) of the blastocyst or from the early epiblast. The cells are truly time, and then follow the success of the transplant over the course pluripotent in the sense that they have the potential to differentiate of months or even years would be a particularly valuable advance. into all of the cell types found in the body and, under appropriate Finally, the ability to provide iPSC from animals with valuable traits culture conditions, to proliferate more or less indefinitely (2, 3). would provide a permanent source of cells for clonal propagation Their discovery provided a new and powerful model for studying that would likely avoid the inefficiencies and problems arising from the programs that drive differentiation and senescence. Crucially, somatic cell nuclear transfer (SCNT), where many of the cloned murine ESC also afforded a means for ‘‘knocking out’’ and ‘‘knock- offspring die or are developmentally abnormal even if they survive ing in’’ genes in mice by homologous recombination, because to term (31). mutated cells with the genetic alteration can be introduced into blastocysts to give rise to chimeric mice that, in turn, can pass on the Results mutated gene through their germline (4, 5). The derivation of Generation of piPSC. Four reprogramming genes (OCT4, SOX2, human ESC from blastocysts, more than 15 years after the first KLF4, and c-MYC), each integrated into separate lentiviral description of ESC in the mouse (6), presaged their recent derivation from other species, including the dog (7), cat (8), and rat (9, 10). Author contributions: T.E., B.P.V.L.T., S. Sachdev, and R.M.R. designed research; T.E., This discovery also heralded the prospects of using ESC in regen- B.P.V.L.T., A.P.A., and S. Sinha performed research; T.E., B.P.V.L.T., and R.M.R. analyzed erative medicine. Despite their undoubted promise as sources of data; and T.E., B.P.V.L.T., and R.M.R. wrote the paper. tissue transplants, many road blocks remain to using human ESC as The authors declare no conflict of interest. a source of transplant material, especially as a means to test the Freely available online through the PNAS open access option. efficacy of therapies and the safety of the transferred cells in Data deposition: The data reported in this paper have been deposited in the Gene animals whose anatomy and physiology better resemble the human Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE15472). than the mouse (11–15). The pig is a potentially useful model in this 1To whom correspondence should be addressed. E-mail: [email protected]. regard because of similarities in organ size, immunology, and whole This article contains supporting information online at www.pnas.org/cgi/content/full/ animal physiology (16–18). 0905284106/DCSupplemental.

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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 Ϸ3 weeks after viral infection. (C) A representative piPSC colony after serial passage resembling hESC shown at low magnification, and (D) at a higher magnification. (Scale bar: A–C, 500 ␮m; D,50␮m.) (E) The piPSC colonies express alkaline phosphatase (AP), and display (F) nuclear localization of OCT4 (green) and surface SSEA1 (red). (Scale bar: 100 ␮m.) (G) Some of the piPSC colonies have a disposition for spontaneous differentiation as evi- denced by the area (arrow) on the right side of the colony. The differentiated cells exhibit cobblestone morphology with a relatively low nucleus to cyto- Fig. 2. Gene expression analysis of piPSC. (A) RT-PCR analysis for expression plasm ratio (Scale bar: 500 ␮m). 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 pc-MYC and pKLF4 showed cross-reactivity. (B) vectors, were introduced into porcine fetal fibroblasts (PFF) and Hierarchical clustering of microarray data for 3 piPSC lines (IC1, ID4 and ID6), and 2 PFF cells (1 and 2) by using Pearson-centered single-linkage rule. All of PFF expressing enhanced green fluorescent protein (EGFP) the genes (n ϭ 8,015) that displayed a fold-change of Ն1.3 of their normalized (Fig. 1A and Fig. S1). Twenty-two days after transduction, expression between piPSC and PFF and a P value of Յ0.05 were used in the compact colonies comprised of cells, which were positive for analysis. The values next to the branches represent Pearson distances. (C) Fold alkaline phosphatase (AP), stage-specific embryonic antigen differences (Log2) in expression of select genes between the piPSC and PFF. Black (SSEA)1, and OCT4, were noted (Fig. 1 B–F). Individual bars on the right-hand side of the axis represent those genes that were up- colonies were mechanically dissociated by using a pulled Pasteur regulated in piPSC compared with PFF, whereas the gray bars on the left side of pipette. A colony’s component cells were transferred to fresh the axis were down-regulated genes. *, P value Յ 0.05 and **, P value Յ 0.01. medium in 24-well plates coated with irradiated MEF. After Ϸ4 days, well-developed secondary colonies (Fig. 1C), resembling (WPRE) region of the lentiviral vector (Table S1). PCR of reverse- hESC colonies, formed. The cells exhibited a high nuclear to cytoplasm ratio with prominently visible nucleoli (Fig. 1D). This transcribed piPSC RNA with these primers clearly showed that all protocol was used to isolate a large number of clonal lines (65 4 human transgenes continued to be coexpressed along with their porcine orthologs in reprogrammed piPSC (Fig. S2A). 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 differenti- Microarray Analysis. Transcript profiling comparisons that used ation, became evident within some colonies, particularly if the Affymetrix porcine microarrays (GEO accession GSE15472) cells were not passaged within 5 days (Fig. 1G). were conducted to provide further confirmation that reprogram- All of the cell lines grew at similar rates, requiring subculture ming of PFF had occurred and to determine the degree of at a roughly 1:10 ratio every 4–5 days. The ID6 line, the only one similarity between different piPSC lines. Hierarchical clustering to be carefully examined, showed a doubling time of Ϸ17 h. of all of the 8,015 genes that were either up- or down-regulated with a P value of Յ0.05 showed that the 3 piPSC lines clustered Expression of Pluripotent Genes in piPSC. Several genes associated together in terms of their overall gene expression and were with pluripotency and known to be expressed in the ICM of pig well-separated from PFF lines (Fig. 2B), although one of the embryos, e.g., OCT4, SOX2, TDGF1 (CRIPTO) and TERT (32, piPSC lines, ID6, appeared as a relative outlier compared with 33), were expressed only in the piPSC, and not in the founder the other two. Of these genes, 2,605 (out of a total of 20,201 PFF (Fig. 2), providing evidence that reprogramming into a represented on the array) displayed at least a 2-fold and 4,297 at pluripotent state had been successful. Results with NANOG least a 1.3-fold increase in expression (P Յ 0.05), whereas over were more equivocal, because expression was also observed in 3,700 genes displayed significantly decreased expression in the the PFF. Nevertheless, as indicated by semiquantitative RT- piPSC relative to the PFF. PCR, NANOG was up-regulated in the putative piPSC relative Several genes associated with the pluripotent state in mouse to the PFF (Fig. 2), again consistent with reprogramming. c-MYC and human pluripotent cells, including SOX2 and NANOG, were and KLF4 expression was detected in the 3 piPSC lines exam- either not represented on the pig array or not annotated. ined, but again both genes were also prominently expressed in However, the regulation of many other genes associated with the PFF (Fig. 2A). Assessments of c-MYC and KLF4 expression pluripotency could be assessed from the microarray data. The were complicated by the fact that the primers designed to amplify log2 fold changes in expression values for these genes are shown the porcine transcripts were also able to detect human c-MYC in Fig. 2C. As anticipated, the majority of such genes were and KLF4 transcripts in hESC (Fig. 2A) and potentially in the up-regulated in the piPSC relative to the PFF. These genes piPSC, if the reprogramming genes continued to be expressed. included CDH1, PODXL, LIN28, GCNF, TNAP, GNL3, CD9, To test this possibility and to distinguish between the pig and ZFP42, UTF1, and, as demonstrated in the previous section, human transcripts, we designed forward primers specific for the OCT4. However, c-MYC and KLF4 were down-regulated in human genes and a common reverse primer corresponding to the piPSC relative to PFF (Fig. 2C; Ϸ97% and 75%, respectively), woodchuck hepatitis virus post-transcriptional regulatory element data that are consistent with the RT-PCR analyses (Fig. 2A). In

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905284106 Ezashi et al. Downloaded by guest on September 25, 2021 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. 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).

Fig. 3. Immunofluorescence staining of pluripotent markers. The immuno- Karyotype Analysis. One of the potential roadblocks in using iPSC fluorescence staining of pluripotent markers OCT4 (A Upper), NANOG (B for therapeutics is acquisition of chromosomal abnormalities. Of Upper), and SOX2 (C Upper) in piPSC colonies cultured on MEF are shown. (A–C the 2 piPSC lines so far subjected to cytogenetic analysis, one Lower) Specific localization to nuclei was confirmed by staining with DAPI (ID6) exhibited a normal karyotype in 19 of 20 cells at passage (blue). 18, whereas the other (IIIC6) had a paracentric inversion in chromosome 16 in 19 of 20 metaphase spreads (Fig. S4), a contrast to c-MYC, mRNA for MYCN was barely detectable in change that was not evident in the original PFF (Fig. S4). PFF, but displayed a 102-fold up-regulation in piPSC. Teratoma Formation. To test for pluripotency in vivo, we trans- Immunocytochemistry. piPSC display many of the same protein planted piPSC (clone ID6) s.c. into dorsal flanks of immunode- ficient (CD-1 nude) mice (n ϭ 2). Three months after injection, and carbohydrate antigens as mouse and human ESC. As Ϸ ϫ anticipated from RT-PCR and microarray data, antibodies one mouse had developed a small, solid tumor 2 3 mm in size against OCT4, NANOG, and SOX2 reacted positively with the that on histological examination was found to be minimally

differentiated. The second mouse had developed several larger, SCIENCES cells of piPSC colonies (Fig. 3 and SI Materials and Methods) and solid tumors lodged within its peritoneal cavity, possibly because negatively with the progenitor PFF cells (Fig. S3B). The cells of poor placement of the injection needle. These tumors con- APPLIED BIOLOGICAL were also positive for the cell surface marker SSEA-1, but tained various kinds of tissue (Fig. 6), including neural epithe- displayed very weak to negative staining for SSEA-3, SSEA-4, lium (ectoderm), striated muscle (mesoderm), and epithelium TRA-1–60, and TRA-1–81 (Fig. S3). with brush border (endoderm). The tumors also included neural fiber (ectoderm), connective tissue, and adipose-like tissue Telomerase Activity of piPSC. All of the 5 piPSC lines tested so far (mesoderm) (Fig. S5). RT-PCR confirmed that these teratomas expressed comparable amounts of telomerase (TERT) activity were of porcine and not murine origin (Fig. S5). Nude mice (n ϭ (Fig. 4), a hallmark of mESC (34). The activity in the piPSC was 2) injected with a comparable number of PFF cells did not at least 5-fold higher than in the hESC line (H9), whereas develop tumors. expression was barely detectable above background in the founder PFF and the feeder MEF cells (Fig. 4). Discussion Since the initial generation of murine iPSC (26), there have been Embryoid Body (EB) Formation and Real-Time RT-PCR Analysis. We numerous follow-up papers describing iPSC from both mouse tested whether piPSC could form EB on culture in the absence and human cells. These studies have used both embryonic and of FGF2 on a nonadhesive substratum. Nine piPSC lines were adult cells and different combinations of reprogramming genes examined, and all, including the three analyzed in Fig. 2, (23, 25–26, 29–30, 35, 37–41). More recently, iPSC have been differentiated into EB under such conditions. When these EB generated from monkey (42), rat (43), and, while this manuscript were subsequently placed on a gelatin coated surface and was under review, miniature swine (44). This technology might cultured in presence of either BMP4, FBS, or RA for 4 additional 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 Ϸ100 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 Fig. 4. Telomerase activity in piPSC. Telomerase activities in piPSC lines (IC1 with human and mouse, a feeder layer of irradiated MEF was 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 essential for maintenance of stem cell properties, but, unlike the samples with 0.2 ␮g of total cell protein and by employing TRAPESE- RT mouse, this requirement could not be substituted by the addition Telomerase Detection Kit (Chemicon). The value, amole, is telomerase activity of LIF to the medium. The lack of LIF dependency is under- assessed by the amount of extended primers with telomeric repeats. The data scored by the down-regulation of LIF receptor gene, LIFR, are presented as means Ϯ SE. during reprogramming (GEO accession GSE15472). As with

Ezashi et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 25, 2021 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 5% FBS for 9 days. (B) Real-time RT-PCR analysis of relative transcript concentrations of pluripotent and lineage-speciﬁc genes in piPSC lines (IC1, ID4, and ID6), PFF, and piPSC differentiated into EB by using either BMP4, FBS, or retinoic acid (RA). The y axis represents fold change relative to GAPDH expression.

human cells, factors produced by MEF in combination with their resemblance to ESC. Another feature common to piPSC FGF2 may be needed to maintain pluripotency and prevent and human and murine ESC is their relatively rapid rate of differentiation. On the other hand, the display of surface marker proliferation. The calculated 17-h linear-phase doubling time is SSEA-1 and the absence of SSEA-3 and -4, are features of slightly greater than that of mouse (50) ESC and roughly similar murine iPSC and ESC and not of human pluripotent cells. The to that of human (51). piPSC also lack TRA-1-60 and TRA-1-81, which are character- Pluripotent stem cells can extend their growth through many istic of human cells (46, 47). The expression of SSEA-1 is doublings without signs of senescence (52). Although the ma- consistent with reports showing this antigen to be expressed on jority of our cell lines have not been taken beyond a few passages, cells of ICM of d 7 pig blastocysts (13, 22, 32, 48) and primordial line ID6, which demonstrates a consistent stem cell-like pheno- germ cells of d 18 to 26 conceptuses, which also lack SSEA-3, type, a normal karyotype, and telomerase activity higher than the human ESC line, H9 (Fig. 4 and Fig. S4), has been subcul- SSEA-4, TRA-1-60, and TRA-1-81 (48, 49). Together, these Ϸ data are consistent with the pluripotent nature of piPSC and tured 40 times at a 1:10 passage ratio at roughly 5-day intervals, which represents at least Ϸ224 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 Fig. 6. Histological section of a solid, encapsulated tumor removed from the patterns of gene expression, despite the complications arising 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, from the incomplete annotation of the porcine array (GEO including neural epithelium (Left, ectoderm), striated muscle (Middle, meso- accession GSE9510, 10469, and 15472). In addition, there was derm), epithelium with brush border (Right, endoderm). Magniﬁcation is up-regulation of expression of other genes, which, although not identical for all 3 tissues. Inset on Right is at a higher magniﬁcation to illustrate categorized as specific to pluripotent cells, are associated with brush border (red arrow) (Scale bar, 5 ␮m). reprogramming of human and murine iPSC (Fig. S6) (54).

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905284106 Ezashi et al. Downloaded by guest on September 25, 2021 Further evidence that the PFF had been efficiently repro- neChip Expression 3Ј-Amplification IVT Labeling Kit (Affymetrix), according to grammed is the very low expression of the differentiation- manufacturer’s instructions. Biotin-labeled cRNA was fragmented and hybrid- associated, fibroblast-specific gene, THY1, in piPSC, which is ized to the Affymetrix GeneChip Porcine Genome Array. Microarrays were rapidly down-regulated when murine fibroblasts are repro- scanned (GeneChip Scanner 3000; Affymetrix), and data were assembled by grammed (54). To our surprise, porcine c-MYC and KLF4 were MicroArray Suite 5.0 software (Affymetrix). The output data were analyzed with GeneSpring GX analysis software (Agilent). The CEL files for each array down-regulated, whereas MYCN was strongly up-regulated in were normalized, and false discovery rates were calculated by T test unpaired, piPSC compared with PFF (Fig. 2B), but examination of the unequal variance with asymptotic p-values and Benjamini-Hochberg multiple microarray data of others (23, 54) revealed that a similar testing correction options. Fold-changes in pairwise comparison of the piPSC phenomenon has been observed in reprogramming of murine and PFF data were calculated based on the unadjusted data means to obtain and human cells. Finally, pNANOG was up-regulated during a list of 8,015 genes with an acceptable fold-change of 1.3. Clustering was reprogramming, although it was already expressed in the paren- performed by the Pearson-centered similarity measure and single linkage rule. tal PFF cells, an observation consistent with previous reports Because the porcine array remains poorly annotated, additional annotation that NANOG is not a reliable marker for pluripotency in pig (32). was performed by using the information of Tsai et al. (70) (Gene Expression Until recently most iPSC have been generated by using Omnibus accession no. GPL6472) and an annotated file provided by Dr. C. K. Tuggle (Iowa State University, Ames, IA) (36). integrating retroviral vectors, leading in some cases, at least, to the continued expression of the transgenes in the reprogrammed Embryoid Body (EB) Formation. Colonies were detached from the MEF feeder cells (29, 55–59). As the ectopically expressed reprogramming layer by manual dissection and transferred into hESC medium without FGF2 genes may cause imbalance in the pluripotency network and even (differentiation medium) in low attachment plates (Corning). After 5 days of influence the ability of reprogrammed cells to differentiate culture, the EB were transferred onto adherent, gelatin-coated tissue-culture optimally (29, 54, 59), ideally the genes should either not be dishes and were cultured in differentiation medium supplemented with either permitted to integrate in the first place (60–62) or excised once 10 ng/ml BMP4 (R & D Systems), 5% FBS, or 0.5 ␮M all-trans retinoic acid (RA; reprogramming has been completed (59, 63). A more recent Sigma) and harvested for RNA isolation 9 days later. strategy has been to provide the reprogramming proteins them- selves directly to the targeted cell type (64) Because retroviruses Real-Time RT-PCR Analysis. Real-time RT-PCR was performed on RNA extracted were used in our experiments, not unexpectedly, transcripts for from EB (at day 9), undifferentiated piPSC, and PFF to assess the relative the 4 transgenes remained detectable in the piPSC (Fig. S2), just concentrations of genes from the 3 germ layers [AFP and NCSTN (endoderm), CRABP2 (ectoderm), and DES (mesoderm)] as well as pluripotent (OCT4 and as they did in the iPSC established from PFF of miniature swine SOX2), and trophoblast genes (CDX2 and PTI). A reference sample constituted SCIENCES (44). Although such expression is not desirable, an impaired by pooling RNA from all of the samples was also used in the analysis. Three ␮g ability to differentiate was not a feature of iPSC described here, of DNase-treated RNA was used in the first strand cDNA synthesis (qScript APPLIED BIOLOGICAL which were fully capable of generating EB expressing genes cDNA synthesis kit; Quanta Biosciences). Validated primer sets for real-time indicative of the 3 germ layers and trophoblast (Fig. 5B) and of PCR analysis are listed in Table S1. Expression of candidate genes relative to producing highly differentiated teratomas in immune- GAPDH in the test was determined with SYBR Green PCR Master Mix (Applied compromised mice (Fig. 6 and Fig. S5). Nevertheless, if these Biosystems) on an ABI PRISM 7500 Real Time System (Applied Biosystems). The cells are to be tested in pigs to mimic therapeutic applications in conditions for real-time RT-PCR were as follows: 50 °C, 2 min; 95 °C,10 min; human patients, it may be necessary to ensure that transgene 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 expression is eliminated completely from the donor lines used by 75 °C for 1 min. The relative expression ratio of target genes was calculated by using one or other of the strategies described above. ⌬⌬Ct method. The specificity of the PCR products amplified was confirmed by dissociation curve analysis. Materials and Methods Cell Culture and Lentiviral Transduction of Human Factors. The ORFs of the Telomerase Activity Assay. Telomerase activity in 0.2-␮g cell protein from human (h) SOX2,hKLF4, and hc-MYC were cloned into the FUGW lentiviral representative piPSC, MEFs, and HESC was determined by using TRAPEZE RT vector (65), and the hOCT4 cDNA was cloned into pSIN18 Telomerase Detection Kit (Chemicon) with Platinum TaqDNA polymerase cPPt.hEF1a.EGFP.WPRE (66) (Fig. S1). Pseudovirus was produced in human (Invitrogen) according to manufacturer’s instructions. 293FT cells (Invitrogen) by transfection with each lentiviral vector along with the VSV-G envelope (pMD2.G) and packaging vector (psPAX2) (67) by using Teratoma Formation. 106 ID6 cells from a confluent 35-mm dish were injected Lipofectamine and PLUS reagents (Invitrogen). Titered virus (68) was used to s.c. into dorsal flanks of two 4-week-old CD-1 nude mice, Crl: CD-1-Foxn1nu ϫ 5 infect the target cells (PFF or EGFP-PFF; 1 10 cells/35-mm dish). On day 2 (Charles River). Tumors were harvested 3 months after injection, dissected, after infection, the cells were dispersed with trypsin and transferred to 10-cm and fixed in 4% paraformaldehyde. Paraffin-embedded tissue was sectioned plates seeded with irradiated MEF. Subsequently, cells were maintained on a and stained with hematoxylin and eosin. All animal experiments were ap- culture medium standardized for human ESC (69) containing 4 ng/ml human proved by the Institutional Animal Care and Use Committee under Protocol FGF2 (Peprotech). No. 4467. Control animals were injected with an equivalent number of PFF cells. Alkaline Phosphatase (AP) Staining and Immunocytochemistry (ICC). AP staining was performed with the AP detection kit (Chemicon). For immunocytochem- ACKNOWLEDGMENTS. We thank Dr. Randall Prather and Lee Spate for provid- istry, cells were fixed in 2% paraformaldehyde in PBS for 20 min at room ing EGFP-PFF and PFF, Drs. Michal Gropp and Benjamin Reubinoff (Hadassah temperature (RT), washed, and exposed to either 5% goat or donkey serum University Medical Center, Jerusalem, Israel) for the pSIN18.cPPT, (Sigma), 1% BSA (Jackson-Immunoresearch), and 0.1% Triton X-100 (Fisher) in hEF1a.EGFP.WPRE plasmid, Dr. David Baltimore (California Institute of Technol- PBS for 30 min. The cells were then incubated with primary antibody (SI ogy, Pasadena, CA) for the FUGW and VSVG envelope plasmids, Dr. Didier Trono Materials and Methods) overnight at 4 °C. After washing, the cells were (Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland) for the ps- incubated with secondary antibody (SI Materials and Methods). Nuclei were PAX2 packaging plasmid, and Dr. Christopher K. Tuggle (Iowa State University, stained with 6 ng/ml DAPI (Invitrogen). Ames, IA) for information on annotation of porcine EST sequences. Kristin Whit- worth assisted in the microarray analysis and Dr. Kei Kuroki (Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, Missouri) provided his- Transcriptional Profiling by Microarray. RNA from 3 piPSC and 2 PFF lines was tological analysis of the teratomas. This work was supported by National Insti- extracted by using STAT-60 (IsoTex Diagnostics). Ten-␮g of each sample was tutes of Health Grants HD42201 and HD21896 (to R.M.R), and Missouri Life subjected to double-stranded cDNA synthesis and labeling by using the Ge- Sciences Trust Fund Grant 00022147 (to T.E.).

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