In vitro and in vivo study on angiogenesis of porcine induced pluripotent stem cell-derived endothelial cells.

Pluripotent stem cells (PSCs) are a promising source of endothelial cells (ECs) for the treatment of cardiovascular diseases. Since clinical application of embryo stem cells (ESCs) involves issues of medical ethics and risk of immune rejection, induced pluripotent stem cells (iPSCs) will facilitate cell transplantation therapy for the cardiovascular diseases. Swine is identified as an ideal large-animal model for human, because of its similar organ size and physiological characteristics. However, there are very few studies on EC differentiation of porcine iPSCs (piPSCs). In recent study, we provided an efficient protocol to differentiate piPSCs into ECs with the purity of 19.76% CD31 positive cells within 16 days. Passaging of these cells yielded a nearly pure population, which also expressed other endothelial markers such as CD144, eNOS and vWF. Besides, these cells exhibited functions of ECs such as uptake of low-density lipoprotein and formation of tubes in vitro or blood vessels in vivo. Our study successfully obtained ECs from piPSCs via a feeder- and serum-free monolayer system and demonstrated their angiogenic function in vivo and in vitro. piPSC-ECs derivation is not only potential for the autologous cell transplantation and cardiovascular drug screening, but also for the mechanistic studies on EC differentiation and endothelial dysfunction.

Derivation and Characterization of Endothelial Cells from Porcine Induced Pluripotent Stem Cells.

Although the study on the regulatory mechanism of endothelial differentiation from the perspective of development provides references for endothelial cell (EC) derivation from pluripotent stem cells, incomplete reprogramming and donor-specific epigenetic memory are still thought to be the obstacles of iPSCs for clinical application. Thus, it is necessary to establish a stable iPSC-EC induction system and investigate the regulatory mechanism of endothelial differentiation. Based on a single-layer culture system, we successfully obtained ECs from porcine iPSCs (piPSCs). In vitro, the derived piPSC-ECs formed microvessel-like structures along 3D gelatin scaffolds. Under pathological conditions, the piPSC-ECs functioned on hindlimb ischemia repair by promoting blood vessel formation. To elucidate the molecular events essential for endothelial differentiation in our model, genome-wide transcriptional profile analysis was conducted, and we found that during piPSC-EC derivation, the synthesis and secretion level of TGF-ß as well as the phosphorylation level of Smad2/3 changed dynamically. TGF-ß-Smad2/3 signaling activation promoted mesoderm formation and prevented endothelial differentiation. Understanding the regulatory mechanism of iPSC-EC derivation not only paves the way for further optimization, but also provides reference for establishing a cardiovascular drug screening platform and revealing the molecular mechanism of endothelial dysfunction.

Interspecies cell fusion between mouse embryonic stem cell and porcine pluripotent cell.

In the area of stem cell research, fusion of somatic cells into pluripotent cells such as mouse embryonic stem (ES) cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell on the pluripotent state of somatic nucleus. As many other groups, we previously established a porcine pluripotent cell line at a low potential. Therefore, here, we performed experiments to investigate if the fusion with mouse ES cell could improve the pluripotent state of porcine pluripotent cell. Our data showed that resultant mouse-porcine interspecies fused cells are AP positive, and could be passaged up to 20 passages. Different degrees of increases in expression of porcine pluripotent genes proved that pig-origin gene network can be programmed by mouse ES. Further differentiation study also confirmed these fused cells' potential to form three germ layers. However, unexpectedly, we found that chromosome loss and aberrant (especially in porcine chromosomes) is severe after the cell fusion, implying that interspecies cell fusion may be not suitable to study porcine pluripotency without additional supportive conditions for genome stabilization.

A novel chemically defined serum- and feeder-free medium for undifferentiated growth of porcine pluripotent stem cells.

Development and improvement of in vitro culture system supporting self-renewal and unlimited proliferation of porcine pluripotent stem cells (pPSCs) is an indispensable process for the naïve pPSCs establishment. In this study, we modified the previous culture system and attempted to develop a novel chemically defined medium (KOFL) for the establishment of pPSCs. It has been cultured >45 passages with flat colony morphology and normal karyotypes in in vitro environment. These cells exhibited alkaline phosphatase activity and expressed pluripotency markers such as OCT4, SOX2, and NANOG, and also possessed differentiation abilities both in vitro and in vivo, proving by the formation of embryonic bodies and teratomas into three germ layers. Then the cells transfected with a green fluorescent protein (GFP) and the GFP positive cells contribute to the porcine preimplantation embryo development. In addition, these cells maintained long duration under feeder-free condition. In conclusion, our results demonstrated that the pPSCs could be derived from preimplantation porcine embryos in serum-free medium and cultured under the feeder-free condition, providing an effective reference for further optimization of the pPSCs culture system.

A novel long intergenic noncoding RNA indispensable for the cleavage of mouse two-cell embryos.

Endogenous retroviruses (ERVs) are transcriptionally active in cleavage stage embryos, yet their functions are unknown. ERV sequences are present in the majority of long intergenic noncoding RNAs (lincRNAs) in mouse and humans, playing key roles in many cellular processes and diseases. Here, we identify LincGET as a nuclear lincRNA that is GLN-, MERVL-, and ERVK-associated and essential for mouse embryonic development beyond the two-cell stage. LincGET is expressed in late two- to four-cell mouse embryos. Its depletion leads to developmental arrest at the late G2 phase of the two-cell stage and to MAPK signaling pathway inhibition. LincGET forms an RNA-protein complex with hnRNP U, FUBP1, and ILF2, promoting the cis-regulatory activity of long terminal repeats (LTRs) in GLN, MERVL, and ERVK (GLKLTRs), and inhibiting RNA alternative splicing, partially by downregulating hnRNP U, FUBP1, and ILF2 protein levels. Hnrnpu or Ilf2 mRNA injection at the pronuclear stage also decreases the preimplantation developmental rate, and Fubp1 mRNA injection at the pronuclear stage causes a block at the two-cell stage. Thus, as the first functional ERV-associated lincRNA, LincGET provides clues for ERV functions in cleavage stage embryonic development.

RE1-silencing Transcription Factor (REST) Is Required for Nuclear Reprogramming by Inhibiting Transforming Growth Factor ß Signaling Pathway.

Differentiated cells can be reprogrammed by transcription factors, and these factors that are responsible for successful reprogramming need to be further identified. Here, we show that the neuronal repressor RE1-silencing transcription factor (REST) is rich in porcine oocytes and requires for nuclear transfer (NT)-mediated reprogramming through inhibiting TGFß signaling pathway. REST was dramatically degraded after oocyte activation, but the residual REST was incorporated into the transferred donor nuclei during reprogramming in NT embryos. Inhibition of REST function in oocytes compromised the development of NT embryos but not that of IVF and PA embryos. Bioinformation analysis of putative targets of REST indicated that REST might function on reprogramming in NT embryos by inhibiting TGFß pathway. Further results showed that the developmental failure of REST-inhibited NT embryos could be rescued by treatment of SB431542, an inhibitor of TGFß pathway. Thus, REST is a newly discovered transcription factor that is required for NT-mediated nuclear reprogramming.

bFGF signaling-mediated reprogramming of porcine primordial germ cells.

Primordial germ cells (PGCs) have the ability to be reprogrammed into embryonic germ cells (EGCs) in vitro and are an alternative source of embryonic stem cells. Other than for the mouse, the systematic characterization of mammalian PGCs is still lacking, especially the process by which PGCs convert to pluripotency. This hampers the understanding of germ cell development and the derivation of authenticated EGCs from other species. We observed the morphological development of the genital ridge from Bama miniature pigs and found primary sexual differentiation in the E28 porcine embryo, coinciding with Blimp1 nuclear exclusion in PGCs. To explore molecular events involved in porcine PGC reprogramming, transcriptome data of porcine EGCs and fetal fibroblasts (FFs) were assembled and 1169 differentially expressed genes were used for Gene Ontology analysis. These genes were significantly enriched in cell-surface receptor-linked signal transduction, in agreement with the activation of LIF/Stat3 signaling and FGF signaling during the derivation of porcine EG-like cells. Using a growth-factor-defined culture system, we explored the effects of bFGF on the process and found that bFGF not only functioned at the very beginning of PGC dedifferentiation by impeding Blimp1 nuclear expression via a PI3K/AKT-dependent pathway but also maintained the viability of cultured PGCs thereafter. These results provide further insights into the development of germ cells from livestock and the mechanism of porcine PGC reprogramming.

Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming.

Aberrant epigenetic reprogramming is the main obstacle to the development of somatic cell nuclear transfer (SCNT) embryos and the generation of induced pluripotent stem (iPS) cells, which results in the low reprogramming efficiencies of SCNT and iPS. Histone H3 lysine 27 trimethylation (H3K27me3), as a repressive epigenetic mark, plays important roles in mammalian development and iPS induction. However, the reprogramming of H3K27me3 in pig remains elusive. In this study, we showed that H3K27me3 levels in porcine early cloned embryos were higher than that in IVF embryos. Then GSK126 and GSK-J4, two small molecule inhibitors of H3K27me3 methylase (EZH2) and demethylases (UTX/JMJD3), were used to regulate the H3K27me3 level. The results showed that H3K27me3 level was reduced in cloned embryos after treatment of PEF with 0.75 µM GSK126 for 48 h, incubation of one-cell reconstructed oocytes with 0.1 µM GSK126 and injection of antibody for EZH2 into oocyte. Meanwhile, the development of the cloned embryos was significantly improved after these treatments. On the contrary, GSK-J4 treatment increased the H3K27me3 level in cloned embryos and decreased the cloned embryonic development. Furthermore, iPS efficiency was both increased after reducing the H3K27me3 level in donor cells and in early reprogramming phase. In summary, our results suggest that H3K27me3 acts as an epigenetic barrier in SCNT and iPS reprogramming, and reduction of H3K27me3 level in donor cells and in early reprogramming phase can enhance both porcine SCNT and iPS efficiency.

Sox2 is the faithful marker for pluripotency in pig: evidence from embryonic studies.

BACKGROUND: Mammalian first lineage segregation generates trophectoderm (TE) and pluripotent inner cell mass (ICM), which provides an ideal model for studying the mechanisms of maintenance and loss of pluripotency. In mouse, the transcription factor OCT4 restricts to ICM and plays a key role in TE/ICM specification and pluripotent regulatory networks. However, in pig, OCT4 does not restrict to ICM cells, suggesting a different molecular basis in TE/ICM specification and pluripotent regulatory networks. RESULTS: To explore molecular basis of porcine TE/ICM specification and pluripotent regulatory networks, we examined expression pattern of pluripotency factors, including SOX2, REX1, SALL4, ESG1, NANOG, TBX3, LIN28, KLF2, and KLF5, in porcine blastocysts. We found that SOX2 is a faithful pluripotent marker that anchored to the pluripotent cells including embryonic part cells, ICM cells and newly EPI cells along with developmental progress, whereas OCT4 expressed in almost all the cells at the same time. Consistently, analysis of spatiotemporal distribution of SOX2 and the TE marker CDX2 revealed an exclusive expression pattern in D6 blastocysts, whereas no correlation was observed between OCT4 and CDX2 at the same stage. CONCLUSIONS: Our results provide a molecular basis in porcine embryonic patterning and a clue for further studying porcine pluripotent regulatory networks.

Identification and characterization of an oocyte factor required for porcine nuclear reprogramming.

Nuclear reprogramming of somatic cells can be induced by oocyte factors. Despite numerous attempts, the factors responsible for successful nuclear reprogramming remain elusive. In the present study, we found that porcine oocytes with the first polar body collected at 42 h of in vitro maturation had a stronger ability to support early development of cloned embryos than porcine oocytes with the first polar body collected at 33 h of in vitro maturation. To explore the key reprogramming factors responsible for the difference, we compared proteome signatures of the two groups of oocytes. 18 differentially expressed proteins between these two groups of oocytes were discovered by mass spectrometry (MS). Among these proteins, we especially focused on vimentin (VIM). A certain amount of VIM protein was stored in oocytes and accumulated during oocyte maturation, and maternal VIM was specifically incorporated into transferred somatic nuclei during nuclear reprogramming. When maternal VIM function was inhibited by anti-VIM antibody, the rate of cloned embryos developing to blastocysts was significantly lower than that of IgG antibody-injected embryos and non-injected embryos (12.24 versus 22.57 and 21.10%; p < 0.05), but the development of in vitro fertilization and parthenogenetic activation embryos was not affected. Furthermore, we found that DNA double strand breaks dramatically increased and that the p53 pathway was activated in cloned embryos when VIM function was inhibited. This study demonstrates that maternal VIM, as a genomic protector, is crucial for nuclear reprogramming in porcine cloned embryos.

Endothelial cells regulate cardiac myocyte reorganisation through ß1-integrin signalling.

BACKGROUND: In normal hearts, capillaries are densely distributed throughout the myocardial tissue, and the cross-talk between myocytes and capillary endothelial cells plays a pivotal role in regulating cardiac development, maturation and function. Although previous studies have suggested a role for the endothelium in the organisation of nearby cardiomyocytes, the underlying mechanism has yet to be illustrated. METHODS AND RESULTS: Using a transwell coculture system, we studied the paracrine effect of endothelial cells on cardiomyocytes and found that the regulation of cardiomyocyte spatial reorganisation and cytoskeletal dynamics by endothelial cells was coupled with ß1-integrin induction. To determine the role of ß1-integrin in this process, we preincubated myocytes with a ß1-integrin function-blocking antibody before coculture. ß1-integrin blockage abolished myocyte chemotactic activity and inhibited microtubule extension and stress fibre assembly. We further evaluated the therapeutic potential of combined endothelial cell-cardiac myocyte transplantation against ischemic cardiomyopathy in an acute myocardial infarction (AMI) mouse model. The results showed that myocytes and endothelial cells synergistically promoted ischemic myocardial repair, as evidenced by the robust engraftment and migration of implanted cells within the infarcted area, as well as the stimulation of angiogenesis, the attenuation of scar tissue and the improvement of cardiac function. CONCLUSION: Our study demonstrated the necessity of ß1-integrin in the interactions between cardiomyocytes and endothelial cells and presented a novel combined transplantation approach that might hold promise for treating ischemic cardiomyopathy.

Tbx3 and Nr5α2 improve the viability of porcine induced pluripotent stem cells after dissociation into single cells by inhibiting RHO-ROCK-MLC signaling.

Porcine induced pluripotent stem cells (piPSCs) had been reported during the past 5years, but there were few reports on how the cell signaling works in piPSCs. In order to clarify the signaling work that dominated the characteristic difference of two types of piPSCs which were derived from Oct4, Sox2, Klf4 and c-Myc (termed 4F piPSCs) and Oct4, Sox2, Klf4, c-Myc, Tbx3 and Nr5α2 (termed 6F piPSCs) respectively, we performed this study. 4F piPSCs and 6F piPSCs were cultured in medium with or without the ROCK inhibitor Y27632 after dissociating into single cells, the efficiency of a single cell colony and the number of AP positive colonies were assessed. The total RhoA and GTP-bind RhoA were detected in 4F piPSCs and 6F piPSCs before and after digestion into single cells. To explore the relationship between RHO-ROCK-MLC signaling pathway and the two factors Tbx3 and Nr5α2, the 4F piPSCs were infected with lenti-virus Tbx3 and Nr5α2 (termed 4F+TND). Results showed that the viability of cells could be enhanced by Y27632 and the RHO-ROCK-MLC signaling pathway was activated after dissociation into single cells in 4F piPSCs but not in 6F piPSCs. And, the 4F+TND piPSCs could be passaged and keep in high viability after dissociation into single cells, though the morphology of colonies did not change. These results indicated that the Tbx3 and Nr5α2 can improve the viability of piPSCs after dissociation into single cells by inhibiting the RHO-ROCK-MLC signaling pathway. And this provides useful information for establishing porcine pluripotent cells in future study.

SINE-Associated LncRNA SAWPA Regulates Porcine Zygotic Genome Activation.

In mice, retrotransposon-associated long noncoding RNAs (lncRNA) play important regulatory roles in pre-implantation development; however, it is largely unknown whether they function in the pre-implantation development in pigs. The current study aims to screen for retrotransposon-associated lncRNA in porcine early embryos and identifies a porcine 8-cell embryo-specific SINE-associated nuclear long noncoding RNA named SAWPA. SAWPA is essential for porcine embryonic development as depletion of SAWPA results in a developmental arrest at the 8-cell stage, accompanied by the inhibition of the JNK-MAPK signaling pathway. Mechanistically, SAWPA works in trans as a transcription factor for JNK through the formation of an RNA-protein complex with HNRNPA1 and MED8 binding the SINE elements upstream of JNK. Therefore, as the first functional SINE-associated long noncoding RNAs in pigs, SAWPA provides novel insights for the mechanism research on retrotransposons in mammalian pre-implantation development.

Comparing bacterial dynamics for the conversion of organics and humus components during manure composting from different sources.

The study aimed to compare the differences in organic fractions transformation, humus components and bacterial community dynamics during manure composting from different sources, and to identify the key biotic and abiotic factors driving the humification process. Five types of manure [pig manure (PM), cow dung (CD), sheep manure (SM), chicken manure (CM), and duck manure (DM)] were used as raw materials for 30 days composting. The results showed the obvious difference of organic fractions decomposition with more cellulose degradation in CD and SM composting and more hemicellulose degradation in PM and CM composting. Composting of PM and CD contained significantly higher humus fractions than the other composts. Fluorescence spectra indicated that SM composting tended to form structurally stable humic acid fractions, while CM and DM tended to form structurally complex fulvic acid fractions. Pearson correlation analysis showed that humification process of composts in category A (PM, CD) with higher humification degree than category B (SM, CM, and DM) was positively correlated with lignin and hemicellulose degradation. Bioinformatics analysis found that Lysinibacillus promoted the degradation of hemicellulose and the conversion of fulvic to humic acid in the composts of category A, and in category B, Thermobifida, Lactobacillus, and Ureibacillus were key genera for humic acid formation. Network analysis indicated that bacterial interaction patterns had obvious differences in composting with different humus and humification levels.

Synthesis and biological evaluation of scutellarein derivatives as neuroprotective agents via activating Nrf2/HO-1 pathway.

Oxidative stress has been considered as the main factor of neurodegenerative diseases. Activation of the Nrf2/HO-1 pathway, as one of the most crucial endogenous protection systems, was regarded as an effective strategy to against oxidative injury. Here, a series of phosphate esters or phosphonates of scutellarein derivatives were designed, synthesized and evaluated on SH-SY5Y cell lines to examine neuroprotective effects against H2O2 induced damage. Among them, compound 16d exhibited more potent cytoprotective effect than the lead compound scutellarin. Preliminary mechanism studies showed that compound 16d could prevent H2O2 induced neuronal apoptosis, significantly decrease ROS generation, elevate SOD and reduce MDA levels in a dose-dependent manner in SH-SY5Y cell lines. Furthermore, western blot assay disclosed that compound 16d could activate Nrf2, and increase the expression of its downstream genes HO-1 in a concentration-dependent manner, thus displaying potent neuroprotective activity. Overall, these findings demonstrated that compound 16d, as a promising neuroprotective agent, deserved further development.

Serum Metabolomic Analysis of Feline Mammary Carcinomas based on LC-MS and MRM Techniques.

INTRODUCTION: To date, there have been no panoramic studies of the serum metabolome in feline mammary carcinoma. As the first such study, metabolomics techniques were used to analyse the serum of cats with these tumours. Three important metabolic pathways of screened differential metabolites closely related to feline mammary carcinomas were analysed to lay a theoretical basis for further study of the pathogenesis of these carcinomas. MATERIAL AND METHODS: Blood in a 5-8 mL volume was sampled from twelve cats of the same breed and similar age (close to nine years on average). Six were feline mammary carcinoma patients and six were healthy. L glutamate, L alanine, succinate, adenine, hypoxanthine, and inosine were screened as were alanine, aspartate, and glutamate metabolism, the tricarboxylid acid (TCA) cycle, and purine metabolism. Data were acquired with LC-MS non-target metabolomics, multiple reaction monitoring target metabolomics, and multivariate statistical and bioinformatic analysis. RESULTS: Expression of five of the metabolites was upregulated and only inosine expression was downregulated. Up- and downregulation of metabolites related to glycometabolism, potentiation of the TCA cycle, greater content of lipid mobilisation metabolites, and abnormality of amino acid metabolism were closely related to the occurrence of the carcinomas. CONCLUSION: These findings provide a new direction for further study of the mechanisms associated with cat mammary neoplasms.

Derivation of porcine extraembryonic endoderm-like cells from blastocysts.

OBJECTIVES: Extraembryonic endoderm (XEN) cells are isolated from primitive endoderm (PrE) of blastocysts. Just like PrE, XEN cells have the ability to differentiate into parietal endoderm (PE) and visceral endoderm (VE), and therefore, they are useful tools for studying mechanisms of PrE cells development and differentiation. Pig is an ideal model for studying human cardiovascular and metabolic diseases and a potential organ source for allotransplantation, while no XEN cell has been obtained from porcine embryos. MATERIALS AND METHODS: Using a serum-free culture system, we directly derived porcine extraembryonic endoderm-like cells (pXEN-like cells) from day 6-7 blastocysts, which could maintain self-renewal for at least 30 passages. RESULTS: The pXEN-like cells resembled mouse XEN cells with large and flat clone morphology and expressed XEN marker genes but not pluripotent genes. Upon in vitro induction, the cells could differentiate into VE and PE. FGF/MEK signalling was not only essential for the maintenance of pXEN-like cells, but also the induction of pXEN-like cells from porcine embryonic stem (pES) cells. CONCLUSIONS: We directly obtained cell lines with XEN characteristics from porcine embryos for the first time. The cells will be helpful tools for studying embryonic development and cell differentiation, which also represent promising cell sources for human regenerative medicine.

Derivation of endothelial cells from porcine induced pluripotent stem cells by optimized single layer culture system.

Regenerative therapy holds great promise in the development of cures of some untreatable diseases such as cardiovascular diseases, and pluripotent stem cells (PSCs) including induced PSCs (iPSCs) are the most important regenerative seed cells. Recently, differentiation of human PSCs into functional tissues and cells in vitro has been widely reported. However, although porcine reports are rare they are quite essential, as the pig is an important animal model for the in vitro generation of human organs. In this study, we reprogramed porcine embryonic fibroblasts into porcine iPSCs (piPSCs), and differentiated them into cluster of differentiation 31 (CD31)-positive endothelial cells (ECs) (piPSC-derived ECs, piPS-ECs) using an optimized single-layer culture method. During differentiation, we observed that a combination of GSK3ß inhibitor (CHIR99021) and bone morphogenetic protein 4 (BMP4) promoted mesodermal differentiation, resulting in higher proportions of CD31-positive cells than those from separate CHIR99021 or BMP4 treatment. Importantly, the piPS-ECs showed comparable morphological and functional properties to immortalized porcine aortic ECs, which are capable of taking up low-density lipoprotein and forming network structures on Matrigel. Our study, which is the first trial on a species other than human and mouse, has provided an optimized single-layer culture method for obtaining ECs from porcine PSCs. Our approach can be beneficial when evaluating autologous EC transplantation in pig models.

The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells.

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.

Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

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.