A stem cell zoo uncovers intracellular scaling of developmental tempo across mammals.

Differential speeds in biochemical reactions have been proposed to be responsible for the differences in developmental tempo between mice and humans. However, the underlying mechanism controlling the species-specific kinetics remains to be determined. Using in vitro differentiation of pluripotent stem cells, we recapitulated the segmentation clocks of diverse mammalian species varying in body weight and taxa: marmoset, rabbit, cattle, and rhinoceros. Together with mouse and human, the segmentation clock periods of the six species did not scale with the animal body weight, but with the embryogenesis length. The biochemical kinetics of the core clock gene HES7 displayed clear scaling with the species-specific segmentation clock period. However, the cellular metabolic rates did not show an evident correlation. Instead, genes involving biochemical reactions showed an expression pattern that scales with the segmentation clock period. Altogether, our stem cell zoo uncovered general scaling laws governing species-specific developmental tempo.

Generation of Marmoset Monkey iPSCs with Self-Replicating VEE-mRNAs in Feeder-Free Conditions.

The generation and culture of transgene-free induced pluripotent stem cells (iPSCs) from the common marmoset (Callithrix jacchus) present unique challenges due to the fact that the protocols developed for culture of human or mouse pluripotent cells are not sufficiently optimized for this particular monkey species. Here, we describe the procedures for the reprogramming of marmoset fetal fibroblasts to pluripotency with self-replicating mRNAs using a two-step approach, where intermediate primary colonies generated in the first reprogramming step are converted in the second step to iPSCs with customized marmoset culture medium. The resulting iPSCs are free of transgenes and can be maintained in long-term culture in feeder-free conditions.

A piggyBac-based platform for genome editing and clonal rhesus macaque iPSC line derivation.

Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. However, the toolbox for the genetic modification of NHPs is less developed than those for other species like mice. Therefore, it is necessary to further develop and refine genome editing approaches in NHPs. NHP pluripotent stem cells (PSCs) share key molecular signatures with the early embryo, which is an important target for genomic modification. Therefore, PSCs are a valuable test system for the validation of embryonic genome editing approaches. In the present study, we made use of the versatility of the piggyBac transposon system for different purposes in the context of NHP stem cell technology and genome editing. These include (1) Robust reprogramming of rhesus macaque fibroblasts to induced pluripotent stem cells (iPSCs); (2) Culture of the iPSCs under feeder-free conditions even after removal of the transgene resulting in transgene-free iPSCs; (3) Development of a CRISPR/Cas-based work-flow to edit the genome of rhesus macaque PSCs with high efficiency; (4) Establishment of a novel protocol for the derivation of gene-edited monoclonal NHP-iPSC lines. These findings facilitate efficient testing of genome editing approaches in NHP-PSC before their in vivo application.

Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules.

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies; however, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Here, we report the derivation of iPSCs from common marmoset monkeys (Callithrix jacchus) using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fibroblasts with VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC and culture in the presence of small molecule inhibitors CHIR99021 and SB431542, we first established intermediate primary colonies with neural progenitor-like properties. In the second reprogramming step, we converted these colonies into transgene-free pluripotent stem cells by further culturing them with customized marmoset iPSC medium in feeder-free conditions. Our experiments revealed a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either toward the neural lineage or further reprogrammed to pluripotency. These results (1) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (2) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.

A translational cellular model for the study of peritubular cells of the testis.

Testicular peritubular cells (TPCs) are smooth muscle-like cells, which form a compartment surrounding the seminiferous tubules. Previous studies employing isolated human testicular peritubular cells (HTPCs) indicated that their roles in the testis go beyond sperm transport and include paracrine and immunological contributions. Peritubular cells from a non-human primate (MKTPCs), the common marmoset monkey, Callithrix jacchus, share a high degree of homology with HTPCs. However, like their human counterparts these cells age in vitro and replicative senescence limits in-depth functional or mechanistic studies. Therefore, a stable cellular model was established. MKTPCs of a young adult animal were immortalized by piggyBac transposition of human telomerase (hTERT), that is, without the expression of viral oncogenes. Immortalized MKTPCs (iMKTPCs) grew without discernable changes for more than 50 passages. An initial characterization revealed typical genes expressed by peritubular cells (androgen receptor (AR), smooth-muscle actin (ACTA2), calponin (CNN1)). A proteome analysis of the primary MKTPCs and the derived immortalized cell line confirmed that the cells almost completely retained their phenotype. To test whether they respond in a similar way as HTPCs, iMKTPCs were challenged with forskolin (FSK) and ATP. As HTPCs, they showed increased expression level of the StAR protein (StAR) after FSK stimulation, indicating steroidogenic capacity. ATP increased the expression of pro-inflammatory factors (e.g. IL1B; CCL7), as it is the case in HTPCs. Finally, we confirmed that iMKTPCs can efficiently be transfected. Therefore, they represent a highly relevant translational model, which allows mechanistic studies for further exploration of the roles of testicular peritubular cells.

A Fosmid-Based System for the Generation of Recombinant Cercopithecine Alphaherpesvirus 2 Encoding Reporter Genes.

The transmission of Macacine alphaherpesvirus 1 (McHV-1) from macaques, the natural host, to humans causes encephalitis. In contrast, human infection with Cercopithecine alphaherpesvirus 2 (CeHV-2), a closely related alphaherpesvirus from African vervet monkeys and baboons, has not been reported and it is believed that CeHV-2 is apathogenic in humans. The reasons for the differential neurovirulence of McHV-1 and CeHV-2 have not been explored on a molecular level, in part due to the absence of systems for the production of recombinant viruses. Here, we report the generation of a fosmid-based system for rescue of recombinant CeHV-2. Moreover, we show that, in this system, recombineering can be used to equip CeHV-2 with reporter genes. The recombinant CeHV-2 viruses replicated with the same efficiency as uncloned, wt virus and allowed the identification of cell lines that are highly susceptible to CeHV-2 infection. Collectively, we report a system that allows rescue and genetic modification of CeHV-2 and likely other alphaherpesviruses. This system should aid future analysis of CeHV-2 biology.

Establishment of porcine and human expanded potential stem cells.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

Intraoperative loading of calcium phosphate-coated implants with gentamicin prevents experimental Staphylococcus aureus infection in vivo.

Orthopedic device-related infection (ODRI) is a potentially devastating complication arising from the colonization of the device with bacteria, such as Staphylococcus aureus. The aim of this study was to determine if intraoperative loading of a clinically approved calcium phosphate (CaP) coating with gentamicin can protect from ODRI in vivo. First, CaP-coated titanium aluminium niobium (TAN) discs were used to investigate the adsorption and release kinetics of gentamicin in vitro. Gentamicin loading and subsequent release from the coating were both rapid, with maximum loading occurring following one second of immersion, and >95% gentamicin released within 15 min in aqueous solution, respectively. Second, efficacy of the gentamicin-loaded CaP coating for preventing ODRI in vivo was investigated using a CaP-coated unicortical TAN screw implanted into the proximal tibia of skeletally mature female Wistar rats, following inoculation of the implant site with S. aureus. Gentamicin-loading prevented ODRI in 7/8 animals, whereas 9/9 of the non-gentamicin treated animals were infected after 7 days. In conclusion, gentamicin can be rapidly and simply loaded onto, and released from, CaP-based implant coatings, and this is an effective strategy for preventing peri-operative S. aureus-induced ODRI in vivo.

Immortalization of common marmoset monkey fibroblasts by piggyBac transposition of hTERT.

Following a certain type-specific number of mitotic divisions, terminally differentiated cells undergo proliferative senescence, thwarting efforts to expand different cell populations in vitro for the needs of scientific research or medical therapies. The primary cause of this phenomenon is the progressive shortening of the telomeres and the subsequent activation of cell cycle control pathways leading to a block of cell proliferation. Restoration of telomere length by transgenic expression of telomerase reverse transcriptase (TERT) usually results in bypassing of the replicative senescence and ultimately in cell immortalization. To date, there have not been any reports regarding immortalization of cells from common marmoset (Callithrix jacchus), an important non-human primate model for various human diseases, with the use of exogenous human TERT (hTERT). In this study, marmoset fibroblasts were successfully immortalized with transposon-integrated transgenic hTERT and expanded in vitro for over 500 population doublings. Calculation of population doubling levels (PDL) showed that the derived hTERT-transgenic lines had significantly higher proliferation potential than the wild-type fibroblasts, which reached only a maximum of 46 doublings. However, the immortalized cells exhibited differences in the morphology compared with the control fibroblasts and transcriptome analysis also revealed changes in the gene expression patterns. Finally, the karyotypes of all hTERT-transgenic cell lines showed various aberrations such as presence of extra Chromosome 17, isochromosome 21q, or tetraploidy. By single-cell expansion of the least affected monoclonal immortalized line, one sub-clonal line with normal karyotype was established, suggesting the possibility to derive immortal marmoset cells with normal karyotypes. The results of this study are an important step towards the development and optimization of methods for the production of immortalized cells from common marmoset monkeys.

Comparative gene expression profiling of pig-derived iPSC-like cells: Effects of induced pluripotency on expression of porcine endogenous retrovirus (PERV).

BACKGROUND: Porcine induced pluripotent stem cells (piPSCs) offer an alternative strategy in xenotransplantation (XTx). As human endogenous retroviruses (HERV), particularly HERV-K, are highly expressed in natural human stem cells, we compared the expression of porcine endogenous retroviruses (PERV) and retrotransposon LINE-1 (L1) open reading frames 1 and 2 (pORF1 and pORF2) in different piPSC-like cell lines with their progenitors (porcine fetal fibroblasts, pFF). METHODS: Cells reprogrammed via Sleeping Beauty-transposed transcription factors were cultured and analyzed on a custom-designed microarray representing the reference pig genome. Data were complemented by qRT-PCR and reverse transcriptase (RT) assay. RESULTS: The expression profiles revealed that 8515 of 26 967 targets were differentially expressed. A total of 4443 targets showed log2 expression ratio >1, and 4072 targets showed log2 expression ratio less than -1 with 0.05 P-value threshold. Approximately ten percent of the targets showed highly significant expression ratios with log2 ≥4 or ≤-4. Besides this general switch in cellular gene expression that was accompanied by an altered morphology, expression of both PERV and L1 pORF1/pORF2 was significantly enhanced. piPSC-like cells revealed a 10-fold to 100-fold higher transcription of the viral PERV-A and PERV-B envelope genes (env), viral protease/polymerase (prt/pol), and L1 elements. No functional retrovirus could be detected under these conditions. CONCLUSION: Epigenetic reprogramming has functional impact on retrotransposons. Thus, the induction of pig-derived pluripotent cells influences their PERV expression profile. Data emphasize the necessity to focus on animals, which show non-functional endogenous viral background to ensure virological safety.

Pigs expressing the human inhibitory ligand PD-L1 (CD 274) provide a new source of xenogeneic cells and tissues with low immunogenic properties.

BACKGROUND: The programmed cell death-1 (PD-1, CD279)/PD-Ligand1 (PD-L1, CD274) receptor system is crucial for controlling the balance between immune activation and induction of tolerance via generation of inhibitory signals. Expression of PD-L1 is associated with reduced immunogenicity and renders cells and tissues to an immune-privileged/tolerogenic state. METHODS: To apply this concept for clinical xenotransplantation, we generated human (h)PD-L1 transgenic pigs and characterized expression and biological function of the transgene at the cellular level. RESULTS: The hPD-L1 was detected in kidney, heart, and pancreas. In addition, peripheral blood mononuclear cells (PBMC), cultured fibroblasts, and endothelial cells were hPD-L1 positive (hPD-L1+ ). The hPD-L1 levels were increased by the treatment of transgenic cells with human cytokines (eg, TNF-α), suggesting a regulatable mode of transgene expression. Compared to cells from wild-type pigs, hPD-L1+ PBMC had a significantly reduced capacity to stimulate proliferation of human CD4+ T cells. Moreover, fibroblasts from hPD-L1 transgenic pigs were partially protected from cell-mediated lysis by human cytotoxic effector cells. CONCLUSIONS: These data indicate a low immunogenic, immune-protected status of cells from hPD-L1 transgenic pigs. The integration of the hPD-L1 concept into existing multi-transgenic pigs is promising to achieve long-term survival of porcine xenografts in non-human primate recipients.

Mouse iPSC generated with porcine reprogramming factors as a model for studying the effects of non-silenced heterologous transgenes on pluripotency.

Mouse somatic cells can be reprogrammed to pluripotency by the ectopic expression of four pluripotency transcription factors, Oct4, Sox2, cmyc, and Klf4. Usually, silencing of the exogenous reprogramming factors is considered to be essential for complete reprogramming and differentiation. In the vast majority of studies, murine pluripotency transcription factor sequences have been used for the reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSC). The effectiveness of xenogeneic transcription factors in miPSC generation has not yet been investigated in detail. Here, we evaluated transposon-based vectors with four porcine pluripotency factors for their ability to reprogram mouse fetal fibroblasts (MEFs) harboring an Oct4-EGFP reporter construct to pluripotency. Additionally, we examined the effects of the non-silenced heterologous transgenes on the expression levels of key endogenous pluripotency markers and the differentiation capacities of the miPSC. Within 8 days of transfection with porcine reprogramming transcription factors the MEFs acquired typical compact miPSC morphology and upregulated expression of endogenous Oct4 and other critical pluripotency genes. Consequently, the transgenes under the control of the TetO promoter became silenced, while the CAG-controlled constructs were expressed throughout the period of culture. Despite the continuous transgene expression, the CAG-miPSC showed normal morphology and were capable of differentiation into the three primary germ layers in vitro and in vivo. However, the expression levels of important endogenous pluripotency markers, Klf4, c-myc, Rex1, and Utf1, were significantly lower in CAG-miPSC compared with TetO-miPSC with silenced reprogramming cassettes. Surprisingly, the endogenous Oct4 and Sox2 expression levels were not affected by the residual transgene expression. Our results suggest that porcine reprogramming transcription factors are suitable for production of miPSC, but silencing of the heterologous transgenes may be necessary for complete reprogramming to pluripotency.

Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3ß- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells.

Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells.

Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF-derived piPSCs were more successful than their FGF-derived counterparts in the generation of in vitro chimeras and in teratoma formation. When LIF piPSCs chimeras were transferred into surrogate sows and allowed to develop, only their prescence within the embryonic membranes could be detected. Whole-transcriptome analysis of the piPSCs and porcine neonatal fibroblasts showed that they clustered together, but apart from the two pluripotent cell populations of early porcine embryos, indicating incomplete reprogramming. Indeed, bioinformatic analysis of the pluripotency-related gene network of the LIF- versus FGF-derived piPSCs revealed that ZFP42 (REX1) expression was absent in both piPSC-like cells, whereas it was expressed in the porcine inner cell mass at Day 7/8. A second striking difference was the expression of ATOH1 in piPSC-like cells, which was absent in the inner cell mass. Moreover, our gene expression analyses plus correlation analyses of known pluripotency genes identified unique relationships between pluripotency genes in the inner cell mass, which are to some extent, in the piPSC-like cells. This deficiency in downstream gene activation and divergent gene expression may be underlie the inability to derive germ line-transmitting piPSCs, and provides unique insight into which genes are necessary to achieve fully reprogrammed piPSCs. 84: 229-245, 2017. © 2016 Wiley Periodicals, Inc.

Long-Term Culture of Porcine Induced Pluripotent Stem-Like Cells Under Feeder-Free Conditions in the Presence of Histone Deacetylase Inhibitors.

The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a complex process that involves significant epigenetic alterations in the reprogrammed cells. Epigenetic modifiers such as histone deacetylase (HDAC) inhibitors have been shown to increase the efficiency of derivation of iPSCs in humans and mice. In this study, we used three HDAC inhibitors, valproic acid, sodium butyrate, and suberoylanilide hydroxamic acid, together with ascorbic acid, for derivation and long-term feeder-free culture of porcine iPS-like cells. In the absence of exogenous growth factors and/or small molecules, these inhibitors were able to maintain the expression of key pluripotency markers, including genes known to be specific for naive pluripotent state in mouse stem cells, for over 60 passages under feeder-free conditions. Surprisingly, the cells became dependent on HDAC inhibitors for the maintenance of proliferation. Moreover, despite showing successful integration into blastocysts upon injection, the cells were unable to undergo normal differentiation in vitro and in vivo in the form of teratomas. Our results suggest that HDAC inhibitors maintain pluripotency gene expression of porcine iPSC-like cells in long-term culture, but prevent lineage specification, requiring further optimization of culture conditions for porcine iPSC derivation.

Kidneys From α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected From Rejection During Ex Vivo Perfusion With Human Blood.

UNLABELLED: Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the α1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties. METHODS: The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20. Two piglets were analyzed by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and sequencing. The biological function of the genetic modifications was tested in a (51)Chromium release assay and by ex vivo kidney perfusions with human blood. RESULTS: Disruption of the GGTA1 gene by deletion of few basepairs was demonstrated in GGTA1-KO/hHO-1/hA20 transgenic pigs. The hHO-1 and hA20 mRNA expression was confirmed by quantitative reverse-transcription polymerase chain reaction. Ex vivo perfusion of 2 transgenic kidneys was feasible for the maximum experimental time of 240 minutes without symptoms of rejection. CONCLUSIONS: Results indicate that GGTA1-KO/hHO-1/hA20 transgenic pigs are a promising model to alleviate rejection and ischemia-reperfusion damage in porcine xenografts and could serve as a background for further genetic modifications toward the production of a donor pig that is clinically relevant for xenotransplantation.

The small molecule inhibitors PD0325091 and CHIR99021 reduce expression of pluripotency-related genes in putative porcine induced pluripotent stem cells.

Small molecule inhibitors of the mitogen-activated protein kinase kinase (MEK) and glycogen synthesis kinase 3 (Gsk3) have been essential in the establishment and maintenance of embryonic stem cells (ESCs) from rats and from nonpermissive mouse strains. However, conflicting results have been reported regarding their efficacy in the establishment and maintenance of pluripotent stem cells from other species. Here, we investigated the effects of PD0325091 (PD; a MEK inhibitor) and CHIR99021 (CH; a Gsk3ß inhibitor) on the reprogramming of porcine fetal fibroblasts to induced pluripotent stem cells (piPSCs). Primary cultures treated with the two inhibitors (2i) showed a reduced number of alkaline phosphatase-positive colonies and a lower percentage of OCT4-expressing cells compared with the cultures grown with basic medium, which was supplemented with murine leukemia inhibitory factor (LIF). Moreover, the piPS-like cell lines established under 2i conditions expressed significantly lower levels of pluripotency markers, including OCT4, SOX2, REX1, UTF1, STELLA, TDH, and CHD1, compared with the controls. To test the short-term effects of the small molecule inhibitors, piPS-like cells that had been established in basic culture medium were cultured for five passages in medium supplemented with 2i or PD or CH individually. In accordance with the first experiment, expression levels of most pluripotency genes declined in cultures treated with inhibitors, although the response to each inhibitory molecule varied for the different genes. Results of this study concur with previous reports and cast doubts on the effectiveness of CH and PD in the reprogramming of porcine somatic cells to pluripotency.

Passage number of porcine embryonic germ cells affects epigenetic status and blastocyst rate following somatic cell nuclear transfer.

Epigenetic instability of donor cells due to long-term in vitro culture may influence the success rate of subsequent somatic cell nuclear transfer (SCNT). Therefore, the present study was designed (1) to investigate the epigenetic changes after prolonged culture in vitro of porcine embryonic germ (EG) cells, including differences in expression levels of both DNA methylation and demethylation-related genes and catalyses of histone modifications, and (2) to assess the efficiency of SCNT using EG cells from different passages. Results showed that genes either associated with DNA demethylation including DNMTs and TET1 or genes related to histone acetylation including HDACs were highly expressed in EG cells at higher passages when compared to EG cells at lower passages. In addition, the expression level of H3K27me3 functional methylase EZH2 increased while no changes were observed on H3K27me3 demethylase JMJD3 in relation to passage number. Moreover, the expression levels of both the H3K4me3 methylase MLL1 and the H3K4me3 demethylase RBP2 were increased at high passages. By using lower passage (numbers 3-5) EG cells as donor cells, the SCNT efficiency was significantly lower compared with use of fetal fibroblast donor cells. However, similar blastocyst rates were achieved when using higher passage (numbers 9-12) EG cells as donor cells. In conclusion, the present study suggests that the epigenetic status of EG cells change with increasing passage numbers, and that higher passage number EG cells are better primed for SCNT.

The choice of expression vector promoter is an important factor in the reprogramming of porcine fibroblasts into induced pluripotent cells.

Porcine induced pluripotent stem cells (iPSCs) are an important animal model for development of regenerative therapies in human medicine. To date, the majority of the porcine cell lines with iPSC characteristics have been generated with the use of viral vectors harboring human or mouse reprogramming factors. Here, we report on the use of Sleeping Beauty transposon vectors based on the porcine transcription factor sequences to reprogram porcine fetal fibroblasts into iPSC-like cells. By using different promoters to drive transgenic expression, we show that the efficiency of reprogramming varies with the promoter type. The cells transfected with two different vector systems under the control of doxycycline-induced tet operator (TetO) promoters failed to upregulate essential endogenous pluripotency genes and to maintain stable iPSC morphology, whereas with the Ef1a and CAG promoters the same vectors proved efficient in generating iPSC-like cells with high levels of endogenous pluripotency gene expression that could be maintained long term in vitro. Our results suggest that the choice of expression vector promoters could significantly influence the efficiency of iPSC production from porcine somatic cells.

In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and Yucatan mini pig embryos at Days 20-24 of gestation.

Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled and individual embryos resulted in the successful derivation of putative EGC lines from Days 20 to 24 with high efficiency. RT-PCR showed that gene expression among all 31 obtained cell lines was very similar, and only minor changes were detected during in vitro passaging of the cells. Genome-wide RNA-Seq expression profiling showed no expression of the core pluripotency markers OCT4, SOX2, and NANOG, although most other pluripotency genes were expressed at levels comparable to those of mouse embryonic stem cells (ESC). Moreover, germ-specific genes such as BLIMP1 retained their expression. Functional annotation clustering of the gene expression pattern of the putative EGC suggests partial differentiation toward endo/mesodermal lineages. The putative EGC were able to form embryoid bodies in suspension culture and to differentiate into epithelial-like, mesenchymal-like, and neuronal-like cells. However, their injection into immunodeficient mice did not result in teratoma formation. Our results suggest that the PGC-derived cells described in this study are EGC-like, but seem to be multipotent rather than pluripotent cells. Nevertheless, the thorough characterization of these cells in this study, and especially the identification of various genes and pathways involved in pluripotency by RNA-Seq, will serve as a rich resource for further derivation of porcine EGC.