• cellular reprogramming
• embryonic stem cells
• induced pluripotent stem cells
• porcine pluripotent stem cells
• transgene-free

• HES7
• Notch signaling
• cellular reprogramming
• developmental allochrony
• segmentation clock
• species-specific developmental timing
• transgene-free porcine iPSC

• Humans
• Animals
• Mice
• Swine
• Induced Pluripotent Stem Cells
• Cellular Reprogramming
• Cell Differentiation
• Transgenes
• Pluripotent Stem Cells
• Mammals

• Farm
• Gene-editing
• New Breeding Technique
• Pigs
• Reproductive Biotechnology
• Sustainability

• ethanol
• feeder cells
• human embryonic stem cells
• human pluripotent stem cells
• self-renewal

• cell biology
• stem cells

• Animals
• Cellular Reprogramming/genetics
• Epigenesis, Genetic
• Fibroblasts/metabolism
• Induced Pluripotent Stem Cells
• Swine
• Swine, Miniature

• P30 CA014520 NCI NIH HHS
• R01 AR064803 NIAMS NIH HHS
• R01 AR064803 NIH HHS
• National Institutes of Health

• cardiac regeneration
• cardiovascular disease
• induced pluripotent stem cells
• large animal models
• myocardial infarction

• PI18/00256 Instituto de Salud Carlos III
• PI17/01487 Instituto de Salud Carlos III
• PIC18/00014 Instituto de Salud Carlos III
• ICI19/00039 Instituto de Salud Carlos III
• PI19/01788 Instituto de Salud Carlos III
• ICI20/00135 Instituto de Salud Carlos III
• RD16/00111/0006 Instituto de Salud Carlos III
• CB16/11/00403 Instituto de Salud Carlos III
• SGR2017 00483 Government of Catalonia
• ND Sociedad Española de Cardiología
• ND Societat Catalana de Cardiologia

• LIN28
• cardiomyocytes
• differentiation
• induced pluripotent stem cells
• pluripotency
• porcine
• reprogramming
• self-renewal

• PGCs
• Porcine
• Regenerative medicine
• Reproduction
• Translational medicine

• Animals
• Cell Differentiation/genetics
• Germ Cells
• Induced Pluripotent Stem Cells
• Pluripotent Stem Cells
• Swine

• cell reprogramming
• fibroblast
• induced pluripotent stem cells
• morphology
• porcine

• DUSP
• LIN28A
• MAPK
• Pluripotency
• piPSCs

• Animals
• Cell Proliferation
• Dual-Specificity Phosphatases/genetics
• Dual-Specificity Phosphatases/metabolism
• Gene Knockdown Techniques
• HEK293 Cells
• Humans
• Induced Pluripotent Stem Cells/physiology
• MAP Kinase Signaling System/physiology
• Mitogen-Activated Protein Kinase Kinases/genetics
• Mitogen-Activated Protein Kinase Kinases/metabolism
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Swine

• avian
• differentiation
• induced pluripotent stem cells
• livestock
• pluripotency
• reprogramming

• Characterization
• Disease modelling
• Disease treatment
• Domestic species
• Induced pluripotent stem cells
• Production
• Veterinary medicine

• Animals
• Animals, Domestic
• Cell Culture Techniques/methods
• Cell Culture Techniques/veterinary
• Cell Differentiation
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/physiology
• Regenerative Medicine/methods
• Veterinary Medicine/methods

Human pluripotent stem cells (hPSCs) represent a promising platform for studying embryonic development, and different states of pluripotency reflect the different stages of embryo development. Here, we successfully converted three in-house-derived primed hPSC lines (H10, H24, and iPS) to a naive state and an expanded pluripotent stem cell (EPS) state. Primed, naive and EPS cells displayed state-specific morphologies and expressed pluripotent markers. The expression of SSEA4 and TRA-1-60 was downregulated in the conversion process. The H3K27me3 expression level also decreased, indicating that global methylation was reduced and that the X chromosome started to reactivate. RNA-sequencing analysis results revealed that differentially expressed genes (DEGs) were significantly enriched in both naive hPSCs and EPS cells when compared to the primed state. However, imprinted gene expression barely changed before and after state reversion. Gene ontology (GO) analyses showed that the upregulated DEGs were mostly enriched in RNA processing, DNA replication and repair, and regulation of cell cycle process, while downregulated DEGs were related to extracellular adhesion and various tissue developmental processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that EPS cells were enriched in the PI3K-Akt and Wnt signaling pathways. Analysis of the lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network between primed, naive hPSCs and EPS cells revealed that hsa-miR-424-5p, has-miR-16-5p, has-miR-27b-3p, has-miR-29c-3p, and KCNQ1OT1 were crucial nodes with high degrees of connectivity. Our work may represent new insight into the intrinsic molecular features of different hPSC states.

• Cells, Cultured
• Down-Regulation
• Embryonic Development/genetics
• Gene Expression Profiling
• Gene Regulatory Networks
• Humans
• Methylation
• MicroRNAs/genetics
• Pluripotent Stem Cells/classification
• RNA, Long Noncoding/genetics
• RNA, Messenger/genetics
• Up-Regulation

• Key Technologies Research and Development Program
• National Natural Science Foundation of China
• Science and Technology Planning Project of Guangdong Province
• Guangzhou Research Collaborative Innovation Projects

• induced pluripotent stem cell
• large animal iPSC
• preclinical animal model

Up until the mid 2000s, the capacity to generate every cell of an organism was exclusive to embryonic stem cells. In 2006, researchers Takahashi and Yamanaka developed an alternative method of generating embryonic-like stem cells from adult cells, which they coined induced pluripotent stem cells (iPSCs). Such iPSCs possess most of the advantages of embryonic stem cells without the ethical stigma associated with derivation of the latter. The possibility of generating “custom-made” pluripotent cells, ideal for patient-specific disease models, alongside their possible applications in regenerative medicine and reproduction, has drawn a lot of attention to the field with numbers of iPSC studies published growing exponentially. IPSCs have now been generated for a wide variety of species, including but not limited to, mouse, human, primate, wild felines, bovines, equines, birds and rodents, some of which still lack well-established embryonic stem cell lines. The paucity of robust characterization of some of these iPSC lines as well as the residual expression of transgenes involved in the reprogramming process still hampers the use of such cells in species preservation or medical research, underscoring the requirement for further investigations. Here, we provide an extensive overview of iPSC generated from a broad range of animal species including their potential applications and limitations.

• Pluripotency
• Embryonic
• Stem cell
• Reprogramming
• Animal
• Wild
• Induced pluripotency

Pigs are important, both for agriculture and as animal models for human diseases. However, due to the lack of embryonic stem cells, the possibility of genetic modification is quite limited. To overcome this limitation, induced pluripotent stem (iPS) cells have been derived from pigs. Despite the public availability of a large number of expression datasets from mice, rats, and primates-derived iPS cells, the expression profile of pig-derived iPS cells is quite limited. Furthermore, there is no dataset focused on the profiling of pig-derived iPS cell with six reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc, Lin28, and Nanog). Here, we used Illumina RNA sequencing platform to characterize the mRNA expression of four-factor derived and six-factor derived pig iPS cells. We observed that the expression levels of whole genes in our established six factors derived iPS cells and parent fibroblast, and compared with that of iPS cells with four factors in public database. These data are valuable in understanding species difference in the reprogramming process of stem cells, and could help identify the key regulating genes involved in the process.

• Induced pluripotent stem cells
• Porcine
• Reprogramming
• Sertoli cells
• Transcription factors

• OTX2
• SOX2
• miR-1343
• piPSCs
• pluripotency

Preparation of mouse embryonic fibroblast (MEF) feeder cells to maintain pluripotent stem cells (PSCs) is time consuming and involved in animal issues. Here, we demonstrated a novel method to prepare feeder cells with high efficiency, timesaving, and low costs. MEFs in 3 × 10⁴ cell/cm² were fixed by methanol for 5 min and air drying for 5 min. Thereafter, the methanol fixed MEF cells (MT-MEF) were able to be used directly to culture PSCs or stored at room temperature for the future usage. PSCs cultured on MT-MEF could be continuously expanded for over 40 passages with the naïve pluripotency. MT-MEFs could also be used to maintain human and pig iPSCs. Moreover, methanol fixed MEFs’ culture dish was able to be reused for at least 4 times, and to be applied for antibiotic resistant screening assay to establishing stable transfected PSC lines. Alternatively, the immortalized cell lines, for instance NIH3T3 cells, could also be fixed by methanol and used as feeder cells to maintain PSCs. Thus, this novel means of methanol fixed feeder cells can completely replace the mitomycin C and gamma radiation treated MEF feeder cells, and be used to maintain PSCs derived from mouse as well as other animal species.

Derivation of bona fide porcine pluripotent stem cells is still a critical issue because porcine embryonic stem cells (ESCs) are not available yet, and most of the culture conditions to maintain porcine induced pluripotent stem cells (piPSCs) are based on conditions for mouse and human iPS cells. In this study, we generated a doxycycline-inducible porcine iPS cell line (DOX-iPSCs) and used it to screen the optimal culture condition to sustain the self-renewal of piPSCs. We found that LIF and b-FGF were required for porcine cell reprogramming, but were not essential cytokines for maintaining the self-renewal and pluripotency of piPSCs. A serum-free 3i medium, which includes three inhibitors CHIR99021, SB431542, and PD0325901, three cytokines BMP4, SCF, and IL-6, and human platelet lysates (PL), was made through serious selections. In 3i condition, the doxycycline-inducible iPSCs could be passaged for a long term without the addition of doxycycline, and the flattened morphology of intermediate state piPSCs could convert to the naïve-like morphology with the increase in endogenous pluripotent gene expressions. Additionally, pPSC cell line isolated from 5.5 days blastocysts could be sustained in 3i medium and the expression of endogenous pluripotent genes OCT4, ESRRB, and STELLA was significantly increased. Our finding directed a new reprogramming strategy by using 3i condition to maintain and convert primed piPSCs into naïve-like pluripotent state. A combination of traditional LIF/b-FGF conditions and 3i condition may help us to find out an appropriate reprogramming approach to generate the naïve state of porcine iPSCs.

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.

• Blastocyst
• ESC
• Embryo
• Epiblast
• Inner cell mass
• Pluripotency
• Stem cells

• ESRRB
• iPS
• pig
• reprograming
• self-renewal

• SSEA-1
• fibroblast
• induced pluripotent stem cell
• porcine
• reprogramming

Previous study showed that expression of epithelial cell adhesion molecule (EpCAM) was significantly upregulated in porcine induced pluripotent stem cells (piPSCs). However, the regulatory mechanism and the downstream target genes of EpCAM were not well investigated. In this study, we found that EpCAM was undetectable in fibroblasts, but highly expressed in piPSCs. Promoter of EpCAM was upregulated by zygotic activated factors LIN28, and ESRRB, but repressed by maternal factors OCT4 and SOX2. Knocking down EpCAM by shRNA significantly reduced the pluripotent gene expression. Conversely, overexpression of EpCAM significantly increased the number of alkaline phosphatase positive colonies and elevated the expression of endogenous pluripotent genes. As a key surface-to-nucleus factor, EpCAM releases its intercellular domain (EpICD) by a two-step proteolytic processing sequentially. Blocking the proteolytic processing by inhibitors TAPI-1 and DAPT could reduce the intracellular level of EpICD and lower expressions of OCT4, SOX2, LIN28, and ESRRB. We noticed that increasing intracellular EpICD only was unable to improve activity of EpCAM targeted genes, but by blocking GSK-3 signaling and stabilizing beta-catenin signaling, EpICD could then significantly stimulate the promoter activity. These results showed that EpCAM intracellular domain required beta-catenin signaling to enhance porcine cell reprogramming.

Pigs are proposed to be suitable large animal models for test of the efficacy and safety of induced pluripotent stem cells (iPSCs) for stem cell therapy, but authentic pig ES/iPS cell lines with germline competence are rarely produced. The pathways or signaling underlying the defective competent pig iPSCs remain poorly understood. By improving induction conditions using various small chemicals, we generated pig iPSCs that exhibited high pluripotency and differentiation capacity that can contribute to chimeras. However, their potency was reduced with increasing passages by teratoma formation test, and correlated with declined expression levels of Rex1, an important marker for naïve state. By RNA-sequencing analysis, genes related to WNT signaling were upregulated and MAPK signaling and TGFβ pathways downregulated in pig iPSCs compared to fibroblasts, but they were abnormally expressed during passages. Notably, pathways involving in DNA repair and replication were upregulated at early passage, but downregulated in iPSCs during prolonged passage in cluster with fibroblasts. Our data suggests that reduced DNA repair and replication capacity links to the instability of pig iPSCs. Targeting these pathways may facilitate generation of truly pluripotent pig iPSCs, with implication in translational studies.

• Activin-SMAD signaling
• NANOG
• iPS cells
• pig
• pluripotency

• Embryonic germ cell
• Handmade cloning
• Induced pluripotent stem cells
• Pluripotency
• Somatic cell nuclear transfer
• Transgenic pigs

The transcription factor Otx2 acts as a negative switch in the regulation of transition from naive to primed pluripotency in mouse pluripotent stem cells. However, the molecular features and function of porcine OTX2 have not been well elucidated in porcine-induced pluripotent stem cells (piPSCs). By studying high-throughput transcriptome sequencing and interfering endogenous OTX2 expression, we demonstrate that OTX2 is able to downgrade the self-renewal of piPSCs. OTX2 is highly expressed in porcine brain, reproductive tissues, and preimplantation embryos, but is undetectable in fibroblasts and most somatic tissues. However, the known piPSC lines reported previously produced different levels of OTX2 depending on the induction procedures and culture conditions. Overexpression of porcine OTX2 can reduce the percentage of alkaline phosphatase-positive colonies and downregulate NANOG and OCT4 expression. In contrast, knockdown of OTX2 can significantly increase endogenous expressions of NANOG, OCT4, and ESRRB, and stabilize the pluripotent state of piPSCs. On the other hand, NANOG can directly bind to the OTX2 promoter as shown in ChIP-seq data and repress OTX2 promoter activity in a dose-dependent manner. These observations indicate that OTX2 and NANOG can form a negative feedback circuitry to regulate the pluripotency of porcine iPS cells.

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

• Animals
• Blastocyst
• Cells, Cultured
• Cloning, Organism
• Diploidy
• Embryo, Mammalian/cytology
• Induced Pluripotent Stem Cells/cytology
• Metaphase
• Mice
• Swine/embryology

• Alternative Splicing/genetics
• Alternative Splicing/physiology
• Animals
• Genetic Variation
• Genome
• Receptors, Estrogen/genetics
• Receptors, Estrogen/metabolism
• Swine/metabolism

Transgenic porcine induced pluripotent stem (iPS) cells are attractive cell sources for the development of genetically engineered pig models, because they can be expanded without senescence and have the potential for multiple gene manipulation. They are also useful cell sources for disease modeling and treatment. However, the generation of transgenic porcine iPS cells is rare, and their embryonic development after nuclear transfer (NT) has not yet been reported. We report here the generation of liver-specific oncogenes (TGF-α/c-Myc)-overexpressing porcine iPS (T/M iPS)-like cells. They expressed stem cell characteristics and were differentiated into hepatocyte-like cells that express oncogenes. We also confirmed that NT embryos derived from T/M iPS-like cells successfully developed blastocysts in vitro. As an initial approach toward porcine transgenic iPS cell generation and their developmental competence after NT, this study provides foundations for the efficient generation of genetically modified porcine iPS cells and animal models.

Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

• Animals
• Blastocyst/cytology
• Cell Culture Techniques
• Cell Differentiation
• Cell Line
• Chimera
• Embryo, Mammalian/cytology
• Embryonic Stem Cells/cytology
• Female
• Fertilization in Vitro/veterinary
• Fibroblasts
• Germ Layers/cytology
• Mice
• Mice, Nude
• Nuclear Transfer Techniques/veterinary
• Pluripotent Stem Cells/cytology
• Pregnancy
• Swine/embryology
• Teratoma

Establishment of embryonic stem cell (ESC) lines has been successful in mouse and human, but not in farm animals. Development of direct reprogramming technology offers an alternative approach for generation of pluripotent stem cells, applicable also in farm animals. Induced pluripotent stem cells (iPSCs) represent practically limitless, ethically acceptable, individuum-specific source of pluripotent cells that can be generated from different types of somatic cells. iPSCs can differentiate to all cell types of an organism’s body and have a tremendous potential for numerous applications in medicine, agriculture, and biotechnology. However, molecular mechanisms behind the reprogramming process remain largely unknown and hamper generation of bona fide iPSCs and their use in human clinical practice. Large animal models are essential to expand the knowledge obtained on rodents and facilitate development and validation of transplantation therapies in preclinical studies. Additionally, transgenic animals with special traits could be generated from genetically modified pluripotent cells, using advanced reproduction techniques. Despite their applicative potential, it seems that iPSCs in farm animals haven’t received the deserved attention. The aim of this review was to provide a systematic overview on iPSC generation in the most important mammalian farm animal species (cattle, pig, horse, sheep, goat, and rabbit), compare protein sequence similarity of pluripotency-related transcription factors in different species, and discuss potential uses of farm animal iPSCs. Literature mining revealed 32 studies, describing iPSC generation in pig (13 studies), cattle (5), horse (5), sheep (4), goat (3), and rabbit (2) that are summarized in a concise, tabular format.

• Cellular reprogramming
• Farm animals
• Induced pluripotent stem cells
• Pluripotency

• Embryonic stem cells
• Gene editing
• Livestock
• Pluripotency
• Transgenic animals

• embryonic stem cells
• embryos
• genetic modification
• induced pluripotent stem cells
• reprogramming

• Embryology
• Embryonic germ cell
• Embryonic stem cell
• Epigenetics
• Induced pluripotent stem cell
• Pluripotency

The domestic pig is an excellent animal model for stem cell research and clinical medicine. There is still no suitable culture condition to generate authentic porcine embryonic stem cells (pESCs) and high quality porcine induced pluripotent stem cells (piPSCs). In this study, we found that culture conditions affected pluripotent and metabolic features of piPSCs. Using defined human embryonic stem cell (hESC) and mouse ESC (mESC) culture conditions, we generated two types of piPSCs, one of which was morphologically similar to hESCs (here called hpiPSCs), the other resembled mESCs (here called mpiPSCs). Transcriptome analysis and signaling pathway inhibition results suggested that mpiPSCs shared more of mESC signaling pathways, such as the BMP pathway and JAK/STAT pathway and hpiPSCs shared more hESC signaling pathways, such as the FGF pathway. Importantly, the mpiPSCs performed embryonic chimera incorporation more efficiently than the hpiPSCs did. In addition, the mpiPSCs showed mitochondrial features of naive ESCs and lipid droplets accumulation. These evidences may facilitate understanding of the gene regulation network and metabolism in piPSCs and promote derivation of bona fide pESCs for translational medicine.

• Animals
• Blastocyst/cytology
• Cells, Cultured
• Embryo, Mammalian/cytology
• Embryonic Development
• Female
• Gene Expression Profiling
• Human Embryonic Stem Cells/cytology
• Humans
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/metabolism
• Lipid Droplets/metabolism
• Lipid Droplets/ultrastructure
• Mice
• Mitochondria/metabolism
• Mitochondria/ultrastructure
• Mouse Embryonic Stem Cells/cytology
• Parthenogenesis
• Signal Transduction
• Sus scrofa
• Transcriptome/genetics

Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals.

• Reprogramming
• Large animal models
• Stemness
• Chimera
• Germline transmission
• Induced pluripotent stem cells
• Gene delivery

• Nr5α2
• ROCK
• Tbx3
• piPSCs

• 3' Untranslated Regions
• Animals
• Biomarkers/metabolism
• Cell Proliferation
• Cell Survival
• Embryonic Stem Cells/cytology
• Embryonic Stem Cells/metabolism
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/metabolism
• Mice
• Swine
• Transcription Factors/genetics
• Transcription Factors/metabolism