This study evaluated the virucidal efficacy of a Bacillus complex composed of Bacillus subtilis YBK-70, Bacillus atrophaeus YBK-W4, and Bacillus circulans YBK-04 (1:1:1, v/v/v) against porcine epidemic diarrhea virus (PEDV). Virucidal efficacy was assessed by measuring the infectivity of PEDV in Vero cells after exposure of the virus to the Bacillus complex culture medium (BCCM) (4×10⁶ colony-forming units (CFU)/mL). PEDV was exposed to the BCCM in the presence of either hard water (HW) or an organic matter suspension (OM). Under HW conditions, PEDV was inactivated by a 50-fold dilution of the BCCM. In the presence of OM, the disinfectant exhibited virucidal activity at a 10-fold dilution. Because BCCM possesses virucidal activity against PEDV, it may serve as an effective reagent for limiting the spread of animal viral diseases.

Background: Endothelial cells (EC) that make up the inner wall of blood vessels play an important role in angiogenesis and vascular recovery. Cardiovascular disease caused by dysfunction of ECs has been reported as a major cause of death worldwide. Despite significant research so far, the underlying mechanism of dysfunction of ECs in cardiovascular disease progression is not yet fully understood. Although therapeutic transplantation of autologous ECs is limited due to lack of cell availability, adiposederived stem cells (ADSCs), known for their ease of procurement and high potential for differentiation, will provide promising solutions to generate autologous ECs. Methods: This study investigated the optimal differentiation of ADSCs into ECs under EBM-2 culture conditions supplemented with VEGF and BMP-4 in hypoxia (2% O2). Results: During 14 days of in vitro differentiation, cells cultured in EBM-2 supplemented with VEGF showed the characteristics of early vascular ECs and some cells adopted polygonal forms. Conversely, cells cultured in EBM-2 and hypoxia supplemented with both VEGF and BMP-4 differentiated into the typical cobblestone morphology that appears in vascular ECs. As a result of immunostaining against the vascular ECs marker CD-31, CD-31 expression was increased under EBM-2 culture conditions with VEGF and VEGF + BMP-4 in hypoxia, but expression was insufficient in normal oxygenation (21% O2). In the flow cytometry analysis, high expression of CD-31 expression was observed under conditions including both VEGF and BMP-4 of hypoxia. Interestingly, in gene expression, the pluripotency marker OCT-3/4 was significantly reduced under hypoxic conditions, but SOX2 and NANOG expression were higher than under normal oxygen conditions. However, CD-31 expression was significantly higher under differentiation conditions in which VEGF and BMP-4 were added under hypoxia conditions. In a functional analysis, CD-31-positive ADSC-derived ECs differentiated under hypoxia had excellent tube formation and Dil-Ac-LDL uptake, which are important for vascular repair and function. Conclusions: These findings confirmed the therapeutic usefulness of ECs derived from ADSC for the treatment of cardiovascular disease due to the synergy effect of hypoxia and BMP-4.

Background: In mammals, DRP1 is a key regulator of mitochondrial fission during mitochondrial dynamics, whereas ATF5 promotes the mitochondrial unfolded protein response (UPRmt). Both pathways are essential for maintaining cellular homeostasis and protecting oocytes and embryos from external stressors. However, the relationship between ATF5 expression and DRP1 under heat stress conditions during porcine oocyte maturation remains unclear. Methods: In this study, we investigated the mitochondrial dynamics and ATF5 expression in porcine oocytes exposed to heat stress during in vitro maturation (IVM). Protein and gene expression levels were assessed using immunofluorescence staining, Western blotting, and quantitative PCR. Results: During IVM, both DRP1 and ATF5 expression were increased (p < 0.01) significantly. In contrast, heat stress markedly impaired (p < 0.05) meiotic progression and cumulus cell expansion. Mitochondrial dynamics were disrupted (p < 0.05), as fission and fusion markers displayed reciprocal changes relative to those in controls. Concomitantly, the expression of ATF proteins was significantly reduced (p < 0.01) under heat stress. Heat-stressed oocytes also exhibited decreased (p < 0.05) expression of genes involved in antioxidant defense and NAD metabolism, whereas autophagy- and apoptosis-related transcripts were significantly upregulated (p < 0.05). At the blastocyst stage, embryos derived from heat-stressed oocytes exhibited nuclear localization of the UPR-associated transcription factors ATF4, CHOP, and ATF5. Conclusions: Collectively, our findings suggest that heat stress disrupts mitochondrial dynamics and ATF5 expression during porcine oocyte maturation while the UPRmt pathway remains active during early embryonic development to mitigate heat-induced cellular damage.

High-mobility group box-1 (HMGB1) is a conserved nuclear protein that stabilizes the nucleosome and regulates gene transcription. Recent studies have reported that glycyrrhizic acid (GA), a HMGB1 inhibitor, blocks extracellular HMGB1 cytokine activity and has a protective effect in various diseases. This study was performed to investigate the effect of GA on in vitro fertilization (IVF) and embryo culture of pig oocytes. HMGB1 was detected by immunofluorescence in the boar sperm post-acrosomal sheath and sperm tail mid-piece, as well as in the nucleus of immature, germinal vesicle stage porcine oocytes, the cytoplasm of mature metaphase II oocytes, and in the embryonic nuclei. A related protein, HMGB2, was also observed in spermatozoa and oocytes, co-localizing with HMGB1. Both HMGB1 and HMGB2 were detected in the protein extracts of spermatozoa and oocytes by Western blotting. Total fertilization rates (mono and polyspermic) increased, and more spermatozoa were bound to the zona pellucida of the oocytes when the IVF medium was supplemented with 20 μM GA compared to the control (p<0.05). In the presence of 20 μM GA, there was a significant increase in the percentage of cleaved embryos, blastocyst formation, and the mean cell numbers per blastocyst (p<0.05). GA treatment increased porcine fertilization rates and improved embryo development in vitro, possibly by blocking the cell-survival-limiting activities of HMGB proteins. Thus, GA could be a suitable therapeutic candidate in assisted reproductive technologies.

This study aimed to develop a powderization strategy using porcine by-products (kidney, liver, and heart) by evaluating the effects of raw material type, pretreatment, and additives (hydroxypropyl methyl cellulose P645 and gelatin) on powder characteristics. Powders from kidney tissue were analyzed for yield, particle structure, compressibility, and size distribution, based on the drying method and additive composition. The spray-dried sample with gelatin at 1:0.5 (w/w) showed 20.4% compressibility and the smallest, most uniform particles, indicating excellent flowability. Due to its superior structural stability, gelatin was selected over HPMC P645. Liver and heart samples that underwent enzymatic hydrolysis and the Maillard reaction were spray-dried with gelatin and assessed for yield and microstructure. The Alcalase-treated liver sample showed the highest yield. Surface analysis confirmed that gelatin formed a protective film enhancing particle stability. These findings suggest gelatin-based spray drying is effective for producing high-quality powders from protein-rich by-products.

Background: The 75-kDa glucose-regulated protein (GRP75) plays a crucial role in regulating the formation of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), facilitating the transfer of Ca2+ ions, and is essential for lipid metabolism, mitochondrial function, calcium homeostasis, and apoptosis in mammalian cells. However, the relationship between GRP75 expression and preimplantation embryonic development in pigs remains unknown. Methods: In this study, we investigated whether GRP75 influences ER–mitochondrial junctions and mitochondrial Ca2+ levels in porcine embryos in vitro . We examined the expression of GRP75 at the zygote, cleavage, and blastocyst stages using immunofluorescence staining and western blot analysis. Results: GRP75 fluorescence and mitochondrial Ca2+ levels were significantly lower (p < 0.01) in the blastocysts than in the zygotes. Western blot analysis revealed a decline in the expression of mitochondrial fusion factors mitofusin 2, GRP75, and the mitochondrial calcium uniporter complex MICU1 protein at the blastocyst stage. To investigate the effects of GRP75 on blastocyst developmental competence, porcinespecific GRP75-siRNA (25 nM) was microinjected at the zygote stage. The results showed a significant decrease in the development capacity until the blastocyst stage (Control: 31.2 ± 2.0%, N.C. siRNA (25 nM): 29.8 ± 3.1%, vs. GRP75-siRNA (25 nM): 24.1 ± 1.6%; p < 0.05). GRP75 in the mitochondria and ER-localized GRP75 were both significantly reduced in blastocysts of pigs microinjected with GRP75 siRNA. Along with ER–mitochondrial colocalization, the MAM formation ratio was significantly reduced in the GRP75-siRNA group compared with that in the control (Control: 29.3% vs. GRP75- siRNA (25 nM): 15.7%, p < 0.001). Conclusions: These findings demonstrate that the GRP75-derived MAM region is involved in the development of early embryos in porcine blastocysts.

Porcine edema disease (ED) is an enterotoxemia of pigs caused by Escherichia coli that produces Stx2e. In this study, the protective efficacy of a recombinant modified Stx2e toxoid was evaluated as a vaccine candidate against ED in piglets. The recombinant Stx2e toxoid was expressed and purified using a commercial E. coli expression system. A total of 25 piglets were used and divided into 5 groups (A to E), with 5 piglets in each group. All piglets (except those in group A) were intramuscularly immunized at 5 days of age (0 weeks post prime immunization; 0 WPPI) and again at 3 weeks of age (2 WPPI). Group B piglets were inoculated with sterile PBS, while groups C to E were immunized with 25 μg/piglet, 50 μg/piglet, and 100 μg/piglet of the recombinant toxoid, respectively. All piglets in groups B to E were orally challenged with virulent wild-type Stx2e⁺ F18⁺ E. coli isolates at 5 weeks of age (4 WPPI). Serum IgG titers in groups D and E were significantly increased from 2 WPPI until the end of the study. Furthermore, no clinical signs were observed in groups A and E during the 7 days following the challenge, while clinical signs of ED were observed in 80%, 60%, and 20% of piglets in groups B, C, and D, respectively. These results indicate that intramuscular vaccination with 100 μg/piglet of the recombinant modified Stx2e toxoid can provide effective protection against ED in piglets.

Background: The cumulus cells (CC) play an essential role in protecting oocytes and providing molecular signals for meiotic and cytoplasmic maturation. Gonadotropins stimulate CC proliferation, promote the release of factors that resume oocyte maturation, and activate small G proteins. Among these, Ras, a GTP-binding protein, participates in signaling pathways that regulate cell growth, division, and proliferation. This study aimed to investigate the changes in the Ras subfamily and gonadotropin receptor expression during porcine CC maturation. Methods: Cumulus-oocyte complexes (COCs) were incubated in a medium supplemented with follicular stimulating hormone (FSH), luteinizing hormone (LH), and epidermal growth factor (EGF) for 44 hours. CCs were collected from the COCs at 0, 22, and 44 hours, and mRNA expression levels of gonadotropin and growth factor receptors (FSHR, LHR, EGFR), Ras subfamily members (H-Ras, K-Ras, N-Ras, R-Ras), and Ras GTPases (RASA1, SOS1) were analyzed using quantitative RT-PCR. Results: The results revealed that LHR and R-Ras mRNA expression significantly increased only at 44 hours compared to the 0-hour group (p < 0.05). Conversely, RASA1 mRNA levels decreased significantly at the same time points. No significant changes were observed in H-Ras, K-Ras, N-Ras , or SOS1 expression. Conclusions: In conclusion, the observed increase in LHR and R-Ras and the decrease in RASA1 provide new insights into the molecular dynamics of Ras subfamily members during porcine CC maturation, contributing to a better understanding of the regulatory mechanisms underlying oocyte development.

Background: Embryo implantation is a complex process regulated by interactions between endometrial epithelial and stromal cells. The endometrium plays a critical role in this process, providing a supportive environment for embryo attachment. However, conventional 2D cell culture models fail to fully replicate the complex 3D structure and cellular interactions of the endometrium. To overcome these limitations, 3D organoid models have been developed to better mimic the in vivo endometrial environment. Methods: In this study, a multicellular uterine organoid model was developed using porcine endometrial epithelial cells (pEECs) and porcine endometrial stromal cells (pESCs) to evaluate the effects of the endometrial environment on embryo implantation. First, single-cell endometrial organoids (pEOs) were formed by culturing pEECs in Matrigel, and their basic cellular characteristics were assessed. Then, a multicellular uterine organoid model was established by combining pEOs with pESCs. Finally, porcine embryos were co-cultured with this model to examine its effect on embryo attachment. Results: The multicellular uterine organoid model facilitated embryo attachment, demonstrating that the 3D structure and cellular interactions of the endometrium play a significant role in embryo implantation. The presence of both epithelial and stromal cells contributed to a more physiologically relevant environment that supported embryo adhesion. Conclusions: This study demonstrates that a multicellular uterine organoid model can serve as a useful in vitro system for porcine embryo implantation research. This model may contribute to a better understanding of embryo development and implantation mechanisms, with potential applications in regenerative medicine and biotechnology.

Background: Pluripotent stem cells (PSCs) are capable of differencing into various cell types in the body, providing them valuable for therapy of degenerative diseases. Patientspecific treatments using PSCs, such as mesenchymal stem cells in patient’s own body, may reduce the risk of immune rejection. Inducing the differentiation of PSCs into vascular endothelial cells (ECs) altering culture conditions or using specific growth factors is able to applied to the treatment of vascular diseases. The purpose of this study was to induce the differentiation of porcine epiblast stem cells (pEpiSCs), bone marrow-derived mesenchymal stem cells (pBM-MSCs) and adipose-derived mesenchymal stem cells (pAMSCs) into ECs and then examine the functionality of vascular ECs. Methods: Porcine pEpiSCs, pBM-MSCs and pA-MSCs were induced to differentiate into ECs on matrigel-coated plates in differentiation medium (EBM-2 + 50 ng/mL of VEGF) for 8 days. Cells differentiated from these stem cells were isolated using CD-31 positive (+) magnetic-activated cell sorting (MACS) and then proliferated in M199 medium. Evaluation of ECs differentiated from these stem cells was treated with capillary-like structure formation and three-dimensional spheroid sprouting assay. Results: Porcine pEpiSCs, pBM-MSCs and pA-MSCs showed similar expression of pluripotency-related genes (OCT-3/4. NANOG, SOX2). These stem cells were differentiated into vascular ECs, but showed different morphologies after the differentiation. Cells differentiated from pEpiSCs showed an elongated spindle-like morphology, whereas cells differentiated from pBM-MSCs showed a round pebble-like morphology. In the case of pA-MSCs, these two morphologies were mixed with each other. Additionally, vascular ECs differentiated from these stem cells showed different formation of capillary-like structure formation and three-dimensional spheroid sprouting assay. Conclusions: Cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs presented the functionality of different vascular ECs, demonstrating the potential of the excellent ECs differentiated from pEpiSCs.

Background: The ability of adeno-associated viruses (AAVs) to transduce various cell types with minimal immune responses renders them prominent vectors for gene editing (GE), with different AAV serotypes exhibiting distinct transduction efficiencies due to their specific cellular tropism. However, detailed molecular processes of AAV infection and penetration, as well as the optimal serotype for specific purposes, remain poorly understood. Porcine models are widely used in research benefitting both human and livestock due to anatomical and physiological similarities to humans. Methods: Transduction efficiencies of 18 AAV serotypes (AAV1–9, 6.2, rh10, DJ, DJ/8, PHP.eB, PHP.S, 2-retro, 2-QuadYF, and 2.7m8) were evaluated in immortalized porcine lung epithelial cells (pLCsImt) and pulmonary alveolar macrophages 3D4/31 (PAMs 3D4/31). Results: We found AAV2, DJ, and 2.7m8 to be the most effective in both cell types. The highest enhanced green fluorescent protein expression of 52.46 ± 2.4% in pLCsImt and 64.08 ± 2.4% in PAMs 3D4/31 was observed for AAV2, while negligible transduction was observed for AAV4, rh10, DJ, PHP.eB, PHP.S, and 2-retro. AAV-DJ showed superior transduction efficiency in PK-15, as compared to AAV2 and 2.7m8. Results emphasize the cell type-specific nature of AAV serotype transduction efficiencies. Notably, AAV2 was most effective in both lung and macrophage cells, whereas AAV-DJ was more effective in renal cells. Conclusions: Our findings suggest that AAV2 was identified as the most efficient serotype for transducing pLCsImt and PAMs 3D4/31, compare to the PK-15 cells. Understanding cell type-specific preferences of AAV serotypes offer crucial insight for tailoring AAV vectors to specific tissue and optimizing genome editing strategies, with potential implications for the advancement of personalized medicine and development of treatments for human and livestock.

Background: Porcine embryonic development is widely utilized in the medical industry. However, the blastocyst development rate in vitro is lower compared to in vivo . To address this issue, various supplements are employed. Extracellular vesicles (EVs) play the role of communicators that carry many bioactive cargoes. Additionally, the contents of EVs can vary on the estrous cycle. Methods: We compared the effects of adding EVs derived from porcine uterine fluid (UF), categorized as non-EV (G1), EVs in estrus (G2) and EVs in diestrus (G3). After in vitro culture (IVC) was performed in three different groups, cleavage rate and blastocyst development rate were examined. In addition, glutathione (GSH) and reactive oxygen species (ROS) levels were measured 2 days after activation to assess oxidative stress. Results: Using NTA and cryo-TEM, we confirmed the presence of EVs with sizes ranging from 30 nm to 200 nm, that the particles were suitable for analysis for analysis. In IVC data, the highest cleavage rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Similarly, the highest blastocyst development rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Conclusions: These results indicate that estrus derived EVs contain biofactors beneficial for early blastocyst development, including GSH which protects the blastocyst from oxidative stress. Additionally, although diestrus-derived EVs are expected to have some effect on blastocyst development, it appeared to be less effective than estrus-derived EVs.

Pathogenic Escherichia coli strains are known to cause edema disease (ED) and postweaning diarrhea (PWD) in piglets. Although the exact mechanisms of pathogenicity that lead to ED-PWD remain to be elucidated, we investigated whether another E. coli adhesin, the plasmid-encoded adhesin involved in diffuse adherence (AIDA) might also be present in ED-PWD-causing E. coli isolates. It was showed that E. coli strains attach to HEp-2 cell assay in three different patterns. Twenty-two isolates were from faeces of preweaned pigs and 36 isolates were from faeces of postweaned pigs. The base sequences for specific oligonucleotide primers of PCR used in this study were constructed based on the regions of conserved sequences between forward (5′-3′) ACAGTATCATATGGAGCCA and reverse (5′-3′) TGTGCGCCAGAACTATTA. Product size was 585 base pairs. A total of 58 AIDA-positive E. coli were used for cell adherence pattern analysis of HEp-2 cell assay. Forty three isolates showed three distinct patterns that were localized adherence (LA), diffuse adherence (DA) and aggregative adherence (AA) patterns respectively. But fifteen isolates were nonadherent. LA (14 isolates) pattern was the most common, followed by DA (13 isolates) and AA (9 isolates) pattern. And three isolates showed a combination of the LA and AA patterns and 4 isolates showed a combination of the DA and AA patterns. Escherichia coli strains that cause nonbloody diarrhea in infants are known to present three distinct patterns of adherence to epithelial cells, namely, localized (LA), diffuse (DA), and aggregative (AA) adherence.

Background: Cadmium (Cd) is toxic heavy metal that accumulates in organisms after passing through their respiratory and digestive tracts. Although several studies have reported the toxic effects of Cd exposure on human health, its role in embryonic development during preimplantation stage remains unclear. We investigated the effects of Cd on porcine embryonic development and elucidated the mechanism. Methods: We cultured parthenogenetic embryos in media treated with 0, 20, 40, or 60 μM Cd for 6 days and evaluated the rates of cleavage and blastocyst formation. To investigate the mechanism of Cd toxicity, we examined intracellular reactive oxygen species (ROS) and glutathione (GSH) levels. Moreover, we examined mitochondrial content, membrane potential, and ROS. Results: Cleavage and blastocyst formation rates began to decrease significantly in the 40 μM Cd group compared with the control. During post-blastulation, development was significantly delayed in the Cd group. Cd exposure significantly decreased cell number and increased apoptosis rate compared with the control. Embryos exposed to Cd had significantly higher ROS and lower GSH levels, as well as lower expression of antioxidant enzymes, compared with the control. Moreover, embryos exposed to Cd exhibited a significant decrease in mitochondrial content, mitochondrial membrane potential, and expression of mitochondrial genes and an increase in mitochondrial ROS compared to the control. Conclusions: We demonstrated that Cd exposure impairs porcine embryonic development by inducing oxidative stress and mitochondrial dysfunction. Our findings provide insights into the toxicity of Cd exposure on mammalian embryonic development and highlight the importance of preventing Cd pollution.

Background: The small intestine plays a crucial role in animals in maintaining homeostasis as well as a series of physiological events such as nutrient uptake and immune function to improve productivity. Research on intestinal organoids has recently garnered interest, aiming to study various functions of the intestinal epithelium as a potential alternative to an in vivo system. These technologies have created new possibilities and opportunities for substituting animals for testing with an in vitro model. Methods: Here, we report the establishment and characterisation of intestinal organoids derived from jejunum tissues of adult pigs. Intestinal crypts, including intestinal stem cells from the jejunum tissue of adult pigs (10 months old), were sequentially isolated and cultivated over several passages without losing their proliferation and differentiation using the scaffold-based and three-dimensional method, which indicated the recapitulating capacity. Results: Porcine jejunum-derived intestinal organoids showed the specific expression of several genes related to intestinal stem cells and the epithelium. Furthermore, they showed high permeability when exposed to FITC-dextran 4 kDa, representing a barrier function similar to that of in vivo tissues. Collectively, these results demonstrate the efficient cultivation and characteristics of porcine jejunum-derived intestinal organoids. Conclusions: In this study, using a 3D culture system, we successfully established porcine jejunum-derived intestinal organoids. They show potential for various applications, such as for nutrient absorption as an in vitro model of the intestinal epithelium fused with organ-on-a-chip technology to improve productivity in animal biotechnology in future studies.

In response to the expanding landscape of the biotechnology industry and the increasing demand for comprehensive drug development as well as the conduct of preclinical and clinical trials, there is a growing need for employment of diverse animal models, including both small and large animals. The focus of this study was on refining ex vivo culture techniques for bioluminescence imaging following administration of intradermal injections in large animals. To examine the feasibility of our approach, varying concentrations of the rFluc protein were administered to rats and live imaging was employed to validate the corresponding levels of expression. Subsequently, following administration of rFluc to mini-pigs, ex vivo analyses were performed on sample tissues to assess the levels of protein expression across different concentrations. In particular, optimal culturing conditions that facilitated the sustained expression of the protein in samples post-euthanasia were identified. Moreover, by employing small animal imaging devices, we were able to capture clear images of the sample plates, which provided evidence of the successful application of our experimental techniques. The findings from this research represent a significant effort toward refining bioluminescence imaging methods tailored for use with large animal models—an imperative facet of contemporary drug development and biomedical research.

Background: Porcine pluripotent stem cells (pPSCs) would provide enormous potential for agriculture and biomedicine. However, authentic pPSCs have not established yet because standards for pPSCs-specific markers and culture conditions are not clear. Therefore, the present study reports comparative pluripotency characteristics in porcine induced pluripotent stem cells (piPSCs) derived from different viral transduction and reprogramming factors [Lenti-iPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM)]. Methods: Porcine fibroblasts were induced into Lenti-iPSCs (OSKM) and Lenti-iPSCs (OSKMNL) by using Lentiviral vector and Sev-iPSCs (OSKM) by using Sendaiviral vector. Expressions of endogenous or exogenous pluripotency-associated genes, surface marker and in vitro differentiation in between Lenti-piPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-piPSCs (OSKM) were compared. Results: Colonial morphology of Lenti-iPSCs (OSKMNL) closely resembles the naïve mouse embryonic stem cells colony for culture, whereas Sev-iPSCs (OSKM) colony is similar to the primed hESCs. Also, the activity of AP shows a distinct different in piPSCs (AP-positive (+) Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but AP-negative (-) LentiiPSCs (OSKM)). mRNAs expression of several marker genes (OCT-3/4, NANOG and SOX2) for pluripotency was increased in Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but Sev-iPSCs (OSKM). Interestingly, SSEA-1 of surface markers was expressed only in Sev-iPSCs (OSKM), whereas SSEA-4, Tra-1-60 and Tra-1-81 were positively expressed in Lenti-iPSCs (OSKMNL). Exogenous reprogramming factors continuously expressed in Lenti-iPSCs (OSKMNL) for passage 20, whereas Sev-iPSCs (OSKM) did not express any exogenous transcription factors. Finally, only Lenti-iPSCs (OSKMNL) express the three germ layers and primordial germ cells markers in aggregated EBs. Conclusions: These results indicate that the viral transduction system of reprograming factors into porcine differentiated cells display different pluripotency characteristics in piPSCs.

Background: Pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer the immense therapeutic potential in stem cell-based therapy of degenerative disorders. However, clinical trials of human ESCs cause heavy ethical concerns. With the derivation of iPSCs established by reprogramming from adult somatic cells through the transgenic expression of transcription factors, this problems would be able to overcome. In the present study, we tried to differentiate porcine iPSCs (piPSCs) into endothelial cells (ECs) for stem cell-based therapy of vascular diseases. Methods: piPSCs (OSKMNL) were induced to differentiation into ECs in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2, EBM-2 + 50 ng/ mL of VEGF) on cultured plates coated with matrigel® (1:40 dilution with DMEM/F-12 medium) for 8 days. Differentiation efficiency of these cells were exanimated using qRT-PCR, Immunocytochemistry, Western blotting and FACS. Results: As results, expressions of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) were higher observed in all porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media than piPSCs as the control, whereas endothelial-associated marker (CD-31) in the differentiated cells was not expressed. Conclusions: It can be seen that piPSCs (OSKMNL) were not suitable to differentiate into ECs in the four differentiation media unlike porcine epiblast stem cells (pEpiSCs). Therefore, it would be required to establish a suitable PSCs for differentiating into ECs for the treatment of cardiovascular diseases.