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cd26 is ubiquitously distributed in the body, particularly in the endothelial and epithelial cells, with the highest expression in the kidney, liver, and small intestine. In humans, cd26 serves as a marker for the embryo implantation phase. However, little is known about the role of cd26 in porcine pre-implantation embryo development. Here, we aimed to examine siRNA-induced cd26 downregulation in the cytoplasm of MII oocytes, to determine whether cd26 is involved in the regulation of porcine pre-implantation embryonic development. The cd26 siRNA was micro-injected into the cytoplasm of MII oocytes, which were then parthenogenetically activated electrically in a medium containing 0.3M Mannitol. Inhibition of the cd26 expression did not affect cleavage but stopped development in the blastocyst stage. Additionally, the cd26 siRNA-treated blastocysts had significantly more apoptotic cells than the untreated blastocysts. Among the 579 transcripts evaluated with transcriptome resequencing, 38 genes were differentially expressed between the treatment and control blastocysts (p < 0.05). Twenty-four genes were upregulated in cd26 siRNA-injected blastocysts, whereas 14 were downregulated. These genes are involved in apoptosis, accumulation of reactive oxygen species, and aberrant expression of ribosomal protein genes. Our results indicate that cd26 is required for proper porcine parthenogenetic activation during embryonic development.

BackgroundTriple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal),N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared betweenalpha-1,3-galactosyltransferasegene-knockout (GTKO) and TKO porcine cells.MethodsPrimary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) fromGTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containingGTKO or TKO pECs was analyzed.ResultsThe C3 deposition level inGTKO pPBMCs or pECs in GVB++was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++-EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar betweenGTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than inGTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence ofGTKO pECs.ConclusionTKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.

Objective: To evaluate the pancreatic differentiation potential of α-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media.Methods: The BM-MSCs have been differentiated into pancreatic β-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media – advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated.Results: The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic β-like cells in the N2B27-based media than in the ADMEM-based media.Conclusion: 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic β-like cells.

Probiotics are defined as live microorganisms that when administered in an appropriate amount, provide health benefits to the host. This study aimed to evaluate the effect of the oral administration of Lactobacillus salivarius (L. salivarius) on growth performance, immunological responses, fecal microbial flora and intestinal mucosal morphology in chickens. Chickens were fed with 109 colony-forming units (CFUs) of wild-type (WT) L. salivarius or phosphate-buffered saline (PBS) for 5 weeks. Chickens body weight was significantly increased by administration of L. salivarius groups compared than control group. The microbial taxonomy in the small intestine and cecum was identified via the chicken feces sample. A total of 286,331 bacterial species were obtained from the chicken fecal samples in overall experimental group. From these, 145,012 bacterial species were obtained from oral administration of L. salivarius treatment group, while 141,319 bacterial species were obtained from control group. Almost 98% of all 16S rRNA sequences from the chicken fecal sample of the two groups were classified into known phyla. Firmicutes, Cyanobacteria, Proteobacteria, Bacteroidetes and Actinobacteria were highly abundant in both groups. Compared with the control birds, the chickens orally administered L. salivarius showed no significant differences in villus length and crypt length. Serum concentrations of the cytokines IL-8, TNF-α, IFN-γ, and IL-4 were markedly reduced in the L. salivarius group. In summary, our findings reveal that L. salivarius can act as a potential probiotic to improve performance and overall gut health in of chickens.

We report a morphometric evaluation of α1,3-galactosyltransferase knockout (GTKO) pig heart and kidney ( n = 9) at the end of one, three and seven months. Organs parameters gradually increased with the age ( p

Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal),Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) fromThe C3 deposition level inTKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.

Background Pigs are considered suitable animal donor models for xenotransplantation. For successful organ transplantation, immune rejection must be overcome. Xenotransplantation has recently been successfully performed using galactose-alpha1,3-galactose epitopes knockout (GalTKO) and a human membrane cofactor protein (hCD46) in a pig model. However, the growth and lifespan of the grafted organ have not been evaluated. Therefore, in the present study we evaluated aging and 84 senescence-related genes using the RT2 Profiler PCR array and whole blood samples from GalTKO/hCD46 Massachusetts General Hospital (MGH) pigs. Methods Experimental groups were double GalTKO/hCD46 (5-month-old), single GalTKO/hCD46 (2-year-old), and non-genetically modified (>3.5-year-old; control group within the same strain). Age-matched white hairless Yucatan (WHY) miniature pig groups were used as controls. Results Among the 19 senescence-related genes selected from the 84 genes for further evaluation, 13 were upregulated in the double GalTKO/hCD46 MGH pigs compared to control MGH pigs; however, in WHY pigs, only 4 genes were up- or down-regulated among the 19 genes. Moreover, in double GalTKO/hCD46 MGH and WHY pigs, the expression of the 19 genes changed only 1- to 2-fold, suggesting that there were no significant differences in senescence signals between the 2 pig lines. Conclusions The present results indicate that the double GalTKO/hCD46 MGH pig might be a suitable model for human xenotransplantation studies. However, we used a limited number of experimental individuals, so further studies using larger experimental groups should be conducted to verify the present results.

Infectious bronchitis (IB) generated by the infectious bronchitis virus (IBV) causes economic difficulties for livestock farmers. The 3D8 single chain variable fragment (scFv) protein is a recombinant antibody with nuclease activity that shows antiviral effects against various DNA and RNA viruses in mice and chickens. In this experiment, 3D8 scFv G2 transgenic chickens produced by crossing 3D8 scFv G1 transgenic rooster and wild type hens were screened by genomic PCR and immunohistochemistry analysis. 3D8 scFv transgenic chickens, wild type sibling chickens, and SPF chickens were directly infected with IBV (5 chickens per group) and indirectly infected by airborne propagation (15 chickens per group). The relative IBV shedding titers were measured by quantitative real-time PCR using oropharyngeal and cloacal swabs on days 3 and 5 after intraocular infection. The viral load was significantly decreased in the 3D8 scFv transgenic chickens from the contact transmission group. Additionally, blood was collected from each group on day 17 post-infection. The ELISA results showed a marked reduction of the antibody titer against IBV in the 3D8 scFv transgenic chickens from the contact transmission group. These results suggest that the 3D8 scFv protein potentially inhibits infectious bronchitis virus transmission in chickens., Highlights • Produced G2 3D8 single chain variable fragment (scFv) transgenic chickens. • 3D8 scFv transgenic chickens showed reduced infectious bronchitis viral shedding level in the contact transmission group. • 3D8 scFv transgenic chickens were 40% lower than the response in the control groups in IBV serum antibody titer.

Background To understand changes in biological responses in nonhuman primate (NHP) recipients of xenotransplantation (XTP), we retrospectively investigated chronological changes in cytokine profiles of NHP recipients after solid-organ XTP. Methods Plasma samples were collected from 7 NHP recipients of pig heart or kidney XTP with α-1,3-galactosyltransferase gene knockout (GTKO) under anti-CD154-based immune suppression at the following time points: immediately before; 2 hours, 3 days, and 7 days after XTP; and weekly thereafter until the graft failed. The plasma levels of the following cytokines were measured: interleukin (IL)-1α, IL-1β, IL-6, IL-12p70, IL-8, IL-10, IL-15, tumor necrosis factor, interferon gamma (IFN-γ), D-dimer, C3a, and histone-complexed DNA fragments. For in vitro experiments, human natural killer (NK) cells were cocultured with wild-type porcine endothelial cells (PECs), GTKO-PECs, and human umbilical vein endothelial cells, with or without anti-CD154 antibody. IFN-γ levels in the culture supernatants were compared. Results IFN-γ levels peaked on day 7 or 10 of XTP and then decreased to basal levels, whereas proinflammatory cytokine levels increased along with the elevation of histone-complexed DNA fragments and were sustained until xenograft failure. In vitro, human NK cells produced more IFN-γ when in contact with wild-type PECs than with human umbilical vein endothelial cells, which was not reduced by the use of GTKO-PECs or addition of anti-CD154 antibody to the mixture. Conclusions In NHP recipients of XTP, the early peak of IFN-γ priming subsequent inflammatory responses may be attributed to NK cell activation in response to xenografts.

Background Recently, mesenchymal stem cells therapy has been performed in dogs, although the outcome is not always favorable. Objectives To investigate the therapeutic efficacy of mesenchymal stem cells (MSCs) using dog leukocyte antigen (DLA) matching between the donor and recipient in vitro. Methods Canine adipose-derived MSCs (cA-MSCs) isolated from the subcutaneous tissue of Dog 1 underwent characterization. For major DLA genotyping (DQA1, DQB1, and DRB1), peripheral blood mononuclear cells (PBMCs) from two dogs (Dogs 1 and 2) were analyzed by direct sequencing of polymerase chain reaction (PCR) products. The cA-MSCs were co-cultured at a 1:10 ratio with activated PBMCs (DLA matching or mismatching) for 3 days and analyzed for immunosuppressive (IDO, PTGS2, and PTGES), inflammatory (IL6 and IL10), and apoptotic genes (CASP8, BAX, TP53, and BCL2) by quantitative real-time reverse transcriptase-PCR. Results cA-MSCs were expressed cell surface markers such as CD90⁺/44⁺/29⁺/45⁻ and differentiated into osteocytes, chondrocytes, and adipocytes in vitro. According to the Immuno Polymorphism Database, DLA genotyping comparisons of Dogs 1 and 2 revealed complete differences in genes DQA1, DQB1, and DRB1. In the co-culturing of cA-MSCs and PBMCs, DLA mismatch between the two cell types induced a significant increase in the expression of immunosuppressive (IDO/PTGS2) and apoptotic (CASP8/BAX) genes. Conclusions The administration of cA-MSCs matching the recipient DLA type can alleviate the need to regulate excessive immunosuppressive responses associated with genes, such as IDO and PTGES. Furthermore, easy and reliable DLA genotyping technology is required because of the high degree of genetic polymorphisms of DQA1, DQB1, and DRB1 and the low readability of DLA 88.

A surrogate eggshell incubation system is a well-defined method to apply to avian genetic modification. In this study, we tried to investigate whether the egg weight differences between donor and surrogate eggs have an effect on donor viability. The groups were divided by egg weight differences between the donor and surrogate eggs into 4 in each system. The viability at d 4 was evaluated at the end of System II, the embryos alive were transferred into the second surrogate eggshells, and the viability at d 5, 6 was evaluated at early phase of System III. Then, the viability of System III was evaluated at different incubation period: d 6-12, d 13-18, d 19-21, and hatching rate was evaluated at d 22. Although the effect of egg weight differences between the donor and surrogate eggs was not observed, a specific group in System III showed higher survival and hatching rate than other group (P0.05).

Probiotics are defined as live microorganisms that when administered in an appropriate amount, provide health benefits to the host. This study aimed to evaluate the effect of the oral administration of Lactobacillus salivarius (L. salivarius) on growth performance, immunological responses, fecal microbial flora and intestinal mucosal morphology in chickens. Chickens were fed with 10

We report a morphometric evaluation of

BACKGROUND: Intestinal organoids have evolved as potential molecular tools that could be used to study host-microbiome interactions, nutrient uptake, and drug screening. Gut epithelial barrier functions play a crucial role in health and diseases, especially in autoimmune diseases, such as inflammatory bowel diseases (IBDs), because they disrupt the epithelial mucosa and impair barrier function. METHODS: In this study, we generated an in vitro IBD model based on dextran sodium sulfate (DSS) and intestinal organoids that could potentially be used to assess barrier integrity. Intestinal organoids were long-term cultivated and characterized with several specific markers, and the key functionality of paracellular permeability was determined using FITC-dextran 4 kDa. Intestinal organoids that had been treated with 2 µM DSS for 3 h were developed and the intestinal epithelial barrier function was sequentially evaluated. RESULTS: The results indicated that the paracellular permeability represented epithelial characteristics and their barrier function had declined when they were exposed to FITC-dextran 4 kDa after DSS treatment. In addition, we analyzed the endogenous mRNA expression of pro-inflammatory cytokines and their downstream effector genes. The results demonstrated that the inflammatory cytokines genes significantly increased in inflamed organoids compared to the control, leading to epithelial barrier damage and dysfunction. CONCLUSION: The collective results showed that in vitro 3D organoids mimic in vivo tissue topology and functionality with minor limitations, and hence are helpful for testing disease models. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-020-00278-0) contains supplementary material, which is available to authorized users.

Background The liver is one of the vital organs involved in detoxification and metabolism. The sex-based differences between the functionality of male and female liver have been previously reported, i.e., male’s liver are good in alcohol clearance and lipid metabolism, while female’s liver are better in cholesterol metabolism. To date, studies on novel drug toxicity have not considered the sex-specific dimorphic nature of the liver. However, the use of hepatocyte-like cells to treat liver diseases has increased recently. Methods Mouse embryos were isolated from a pregnant female C57BL/6J mouse where mouse embryonic fibroblasts (MEFs) were isolated from back skin tissue of each embryo. MEFs were transduced with human transcription factors hHnf1α, hHnf4α, and hFoxa3 using the lentiviral system. The transduced MEFs were further treated with hepatocyte-conditioned media followed by its analysis through RT-qPCR, immunofluorescence, functional assays, and finally whole-transcriptome RNA sequencing analysis. For in vivo investigation, the mouse hepatocyte-like cells (miHep) were transplanted into CCl4-induced acute liver mouse model. Results In this study, we evaluated the sex-specific effect of miHep induced from male- and female-specific mouse embryonic fibroblasts (MEFs). We observed miHeps with a polygonal cytoplasm and bipolar nucleus and found that male miHeps showed higher mHnf4a, albumin secretion, and polyploidization than female miHeps. Transcriptomes from miHeps were similar to those from the liver, especially for Hnf4a of male miHeps. Male Cyps were normalized to those from females, which revealed Cyp expression differences between liver and miHeps. In both liver and miHeps, Cyp 4a12a and Cyp 4b13a/2b9 predominated in males and females, respectively. After grafting of miHeps, AST/ALT decreased, regardless of mouse sex. Conclusion In conclusion, activation of endogenic Hnf4a is important for generation of successful sex-specific miHeps; furthermore, the male-derived miHep exhibits comparatively enhanced hepatic features than those of female miHep.

Probiotics have been defined as live microorganisms that are administered in an appropriate amount to provide health benefits to the host animal. In this study, we investigated the effect of L. salivarius DJ-sa-01 secreting the 3D8 single-chain variable fragment (3D8 scFv) on the growth performance, cytokine secretion, and intestinal microbial flora of chickens. The experiment was divided into the control group and L. salivarius expressing 3D8 scFv experimental group. Chicken was fed 109 colony-forming units (CFUs) of wild-type (WT) L. salivarius or 3D8 scFv-secreting L. salivarius daily for 35 days. The administration of L. salivarius expressing 3D8 scFv significantly improved the body weight of chickens compared with the administration of WT L. salivarius. A 16S ribosomal RNA metagenomic analysis showed that Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla in both experimental groups. At the genus level, Lactobacillus was more abundant (22.82%) in the L. salivarius/3D8 group compared with the WT L. salivarius group. The serum levels of cytokines, such as IL-8, TNF-α, IL-1β, IFN-γ, IL-4, and IGF1, were significantly reduced in the L. salivarius/3D8-treated chickens. In summary, our results suggest that L. salivarius expressing 3D8 scFv could be considered a feed additive for improving the growth performance, immune function, and disease resistance of poultry.

The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted (1.5×108/ml) in lactose egg yolk (LEY) and cooled at 5°C for 3 h. The cooled semen was then diluted (1×108/ml) in LeIBP containing LEY with 9% glycerol and maintained at 5°C for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen (LN2) vapors for 20 minutes and then plunged into LN2. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig (GalT-MCP/-MCP). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain GalT-MCP/-MCP/CD39/CD73 pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from GalT-MCP/-MCP pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate GalT-MCP/-MCP/CD39/CD73 pig expressing CD39 and CD73 at endothelial cells.

Previously, Escherichia coli harboring the codon-optimized 3D8scFv gene (E. coli 3D8scFv) was developed as a feed additive for use in preventing norovirus infection. Here, we evaluated whether the 3D8scFv gene affects the colonization of E coli when E. coli 3D8scFv passes through the mouse gastrointestinal tract. To determine the colonization ability of E. coli 3D8scFv, E. coli cells with or without the 3D8scFv gene were fed to mice. Total DNA was extracted from the animals’ stools, stomach, small intestine and colon. All samples were amplified using 3D8scFv gene-specific primer sets. E. coli 3D8scFv begins to be excreted 1 h after feeding and that all E. coli 3D8scFv cells were excreted between 12 and 24 h after the last feeding of the cells. The previously measured gastrointestinal transit time of the mice was between 8 h and 22 h. The results of this study therefore show that E. coli 3D8scFv cannot colonize the gastrointestinal tracts of mice. In addition, if the purified 3D8 scFv protein is used as a feed additive, any associated E. coli 3D8scFv bacteria will not colonize the gastrointestinal tracts of the livestock. Thus, this feed additive meets the safety assessment criteria for the commercial use of bacteria., Highlights • It is evaluated whether E. coli 3D8scFv colonizes in the gastrointestinal tracts of mice. • Orally ingested E. coli 3D8scFv is excreted from mice without colonization of the gastrointestinal tract. • Purified 3D8 scFv is suitable for a feed additive according to the concept of substantial equivalence.

Tissue engineering (TE) has been developed to create functional organs and tissue by combining 3D matrix and cells in vitro. Vascularization and angiogenesis are utmost important for supply of nutrients and oxygen in tissue engineered organs. The present study was performed to isolate and characterize primary endothelial cells (EC) from aorta of alpha 1, 3-enzyme galactosyltransferase knock out (GalT KO) pig, to minimize immune rejection and analyze body immune system for future xenotransplantation studies. Isolation of primary EC from aorta were performed by incubation with dispase for 8-10 min at 37°C. Primary EC were cultured in EC growth medium on different extra cellular matrix (ECM), either collagen or gelation. Primary EC exhibits morphological characteristics and showed positive expressions of EC specific marker proteins i.e. PECAM1, KDR and VWF despite of their ECM surface; however, on collagen based surface they showed increase in mRNA level analyzed by qPCR. Primary EC cultured on collagen were sorted by flow cytometer using KDR marker and cultured as KDR positive cells and KDR negative cells, respectively. KDR positive cells showed dramatically increased in PECAM1 and VWF level as compared to KDR negative cells. Based on the above results, primary EC derived from GalT KO are successfully isolated and survived continuously in culture without becoming overgrown by fibroblast. Therefore, they can be utilize for xeno organ transfer, tissue engineering, and immune rejection study in future.

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous α 1,3-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig (GalT-MCP/+), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig (GalT-MCP/-MCP) by crossbreeding GalT-MCP/+ pigs. Two female founders gave birth to six of GalT-MCP/-MCP, and seven GalT-MCP/+ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the GalT-MCP/-MCP pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the GalT-MCP/-MCP pig is a useful candidate to apply xenotransplantation study.

Primary hepatocytes (PHs) are considered the ‘gold standard’ in drug screening owing to their ability to express many drug-metabolizing enzymes and transporters. Culturing hepatocytes and maintaining their fate in vitro is a major issue since last decade. The main problem with in vitro hepatocytes culture is that they rapidly lose their hepatic morphology and liver-specific functions in culture. Herein, we isolated rat PHs, and cultured them in monolayers (2-D) and spheroids (3-D). The 2-D-cultured PHs exhibited elongated morphology, whereas the 3-D-cultured PHs exhibited spheroid morphology with gradual diameter decrease until 7 days. After 7 days of in vitro culture, PHs were analyzed for the expression of hepatic (Alb, Tf, and Afp) and apoptotic markers (Bax and Bcl2), and co-expression of CYP3A1 and Abumin after 2 and 7 days. Furthermore, in both cultures, PHs were induced with 3-methylcholanthrene (3-MC, Cyp1a-specific inducer) and dexamethasone (Cyp3a-specific inducer) for 48 and 72 h, respe...

In this study, we attempted to upgrade GT(−MCP/−MCP) pig genetically to express MCP at a higher level and additionally thrombomodulin (TBM), which have respective roles as a complement regulatory protein and a coagulation inhibitor. We constructed a dicistronic cassette consisting of codon-optimized MCP (mMCP) and TBM (m-pI2), designed for ubiquitous expression of MCP and endothelium specific expression of TBM. The cassette was confirmed to allow extremely increased MCP expression compared with non-modified MCP, and an endothelial-specific TBM expression. We thus transfected m-pI2 into ear-skin fibroblasts isolated from a GT(−MCP/−MCP) pig. By twice selection using magnetically activated cell sorting (MACS), and single-cell culture, we were able to obtain clones over 90% expressing MCP. The cells of a clone were provided as a donor for nuclear transfer resulting in the generation of a GT(−MCP/−MCP)/mMCP/TBM pig, which was confirmed to be carrying cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Collectively, these results demonstrated an effective approach for upgrading transgenic pig, and we assumed that upgraded pig would increase graft survival. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-2091-z) contains supplementary material, which is available to authorized users.

The present study aimed to investigate the characteristics of mPEA15 expressing transgenic pig (TG pig) as a potential model for diabetes. Expression analysis confirmed the ubiquitous expression of mPEA15 in TG pigs at F4. Oral glucose tolerance test results showed that restoration of normal glucose levels was significantly delayed in the TG pigs when compared with that in the wild-type pigs (WT pigs). Primary skeletal muscle cells isolated from TG pigs demonstrated reduced glucose uptake and reduced GLUT4 translocation to the plasma membrane in response to insulin treatment. Combined, these results suggest that mPEA15 expressing pigs has a glucose intolerance and insulin resistance which are known to mediate the pathophysiology of type 2 diabetes mellitus. Thus, mPEA15 transgenic pigs would serve as a promising model for diabetes translational research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-019-2021-0) contains supplementary material, which is available to authorized users.

The present study was aimed to investigate the effects of 3D8 scFv-secreting Probiotic Lactobacillus reuteri (L. reuteri) on growth performance, inflammatory responses, and intestinal microbial flora in chickens. To this end, a total of 14 healthy wild-type chickens were divided into two experimental groups. Each group was orally administrated with a daily dose of 109 colony-forming units (CFU) of 3D8 scFv-producing L. reuteri or wild-type (WT) for 35 days. Administration of L. reuteri/3D8 scFv significantly improved the body weight of chickens when compared to L. reuteri/WT group. The bacterial taxonomic composition of the fecal microbiota was determined by pyrosequencing of 16S rRNA gene amplicons. Firmicutes, Actinobacteria, and Proteobacteria were dominant phyla in two experimental groups. However, in 3D8 L. reuteri treatment groups at genus level, the Lactobacillus was highly abundant, being represented by 18.12%. In addition, serum levels of primary cytokines such as IL-6, IL-8, TNF-α, IFN-γ, IL-4, and IGF1 were markedly reduced in the probiotic L. reuteri 3D8 group. In summary, our results indicate that the administration of L. reuteri expressing 3D8 scFv has a modulatory effect on inflammatory responses, improves weight gain while not affecting the common microbial composition of the chicken intestine.

The present study was aimed to investigate the effects of 3D8 scFv-secreting Probiotic Lactobacillus reuteri (L. reuteri) on growth performance, inflammatory responses, and intestinal microbial flora in chickens. To this end, a total of 14 healthy wild-type chickens were divided into two experimental groups. Each group was orally administrated with a daily dose of 10(9) colony-forming units (CFU) of 3D8 scFv-producing L. reuteri or wild-type (WT) for 35 days. Administration of L. reuteri/3D8 scFv significantly improved the body weight of chickens when compared to L. reuteri/WT group. The bacterial taxonomic composition of the fecal microbiota was determined by pyrosequencing of 16S rRNA gene amplicons. Firmicutes, Actinobacteria, and Proteobacteria were dominant phyla in two experimental groups. However, in 3D8 L. reuteri treatment groups at genus level, the Lactobacillus was highly abundant, being represented by 18.12%. In addition, serum levels of primary cytokines such as IL-6, IL-8, TNF-α, IFN-γ, IL-4, and IGF1 were markedly reduced in the probiotic L. reuteri 3D8 group. In summary, our results indicate that the administration of L. reuteri expressing 3D8 scFv has a modulatory effect on inflammatory responses, improves weight gain while not affecting the common microbial composition of the chicken intestine.

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5’-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.

Pigs have been used as a good research model for xenotransplantation. Several groups have generated porcine-induced pluripotent stem cells (piPSCs) from differentiated somatic cells. Transgenic pigs with the alpha1,3-galactosyltransferase gene-knockout (GalT-KO) could successfully govern hyper acute rejection of organ transplants into primates. Thus, GalT-KO piPSCs could be a powerful cell resource for agricultural and biomedical applications. This study was performed to generate iPSCs from GalT-KO pigs and characterize their properties. We successfully generated a GalT-KO iPSC from a genetically modified pig using double alpha1,3-galactosyltransferase knockout alterations. Similar to mouse embryonic stem cells, the GalT-KO piPSCs were positive for classical pluripotency markers: POU5F1, NANOG, SOX2 and SSEA1, and were negative for: SSEA3, TRA-1-60 and TRA-1-81. Furthermore, these cells could form an embryoid body that differentiated into three germ layers in vitro, and were highly proliferative u...

Background: Corneal allotransplantation is a well-known technique to treat corneal blindness. However, the problem of organ scarcity in transplantology has become profound. One promising alternative could be xenograft using pig as an organ donor. Full thickness corneal xenotransplantation needs total immunosuppression. This study is an investigation of the efficacy of α1,3-ga lactosyltransferase gene-knockout (GTKO) transgenic pig-to-nonhuman primate lamellar corneal transplantation with minimal immunosuppression. Methods: We conducted 10 lamellae corneal xenotransplantation between 2016 and 2019. Clinically acceptable graft size (diam eter 7.5 mm, thickness 500 um). The dexamethasone subconjunctival injection (1.5 mg/0.3 mL) was administered for minimal immunosuppression immediately after surgery and eye drops of 0.5% levofloxacin and 1% prednisolone acetate were applied four times a day for 1 week, gradually tapered. No eye drops were added after two months. Three cases are alive without graft rejection. We examined the remaining seven cases for the histopathological features and Immunologic profiles. Results: As a result, three of the ten xenografts survival is significantly longer 1,239, 589, and 316 days. Corneal opacity result ed in graft failure, and terminated in seven cases. Rejected grafts showed extensive polymorphic cellular infiltration, different degrees of epithelial layer irregular attenuation, stromal neovascularization, and inflammatory cell infiltrations including lym phocytes, plasma cells, eosinophils, neutrophils. Stromal irregularity, fibrosis and edema are observed in two of seven cases resulting in a single case of sub epithelial detachment. Immunologic profiles of the recipients with rejected grafts shows minimal increase in anti-Gal antibody IgG and IgM but increase in anti-Gal IgG is seen in one case. Four cases have different systemic in flammatory conditions with regards to plasma C3a and D-dimer levels. The anterior stromal surface of the graft showed epitheli al nests and fibrous proliferation. Conclusions: The GTKO transgenic pig to NHP lamella xeno corneal transplantation could be a promising substitute for human corneal allograft. Lamella xeno corneal transplantation may be a feasible option with minimal immunosuppression.

The anti-inflammatory effects of oridonin (Ordn) have been well established in previous studies. However, the apoptotic effects of Ordn on oral cancer cells have not yet been evaluated, at least to the best of our knowledge. The aim of this study was to examine the apoptotic activity of Ordn in oral squamous cell carcinoma cells and to eluciudate the underlying mechanisms. For this purpose, we employed experimental techniques, such as MTT assay, DAPI staining, soft agar assay, flow cytometry and western blot analysis. Our results revealed that Ordn suppressed oral cancer cell proliferation and soft agar colony formation, while it induced reactive oxygen species (ROS)-dependent apoptosis in a dose or time-dependent manner. The generation of ROS was detected in HN22 and HSC4 cells treated with Ordn and the use of the free radical scavenger, N-acetyl-L-cysteine, almost blocked Ordn-induced apoptosis. The phosphorylation of JNK and p38 mitogen-activated protein kinase (MAPK) was manifested in the Ordn-treated cells. Furthermore, Ordn induced the apoptosis of oral cancer cells through the mitochondrial-dependent pathway, involving the loss of mitochondrial membrane potential, the release of cytochrome c, the induction of poly(ADP-Ribose) polymerase (PARP) cleavage, alterations in the ratios of apoptotic proteins and the activation of the caspase cascade. Taken together, these findings indicate that Ordn induces the apoptosis of oral cancer cells probably via ROS-mediated JNK/p38 MAPK and mitochondrial pathways; thus, Ordn may have potential for use in the treatment of oral cancer.

Background Licochalconce (LC) H is an artificial compound in the course of synthesizing LCC in 2013. So far, few studies on the effects of LCH have been found in the literature. Despite progress in treatment modalities for oral cancer, the cure from cancer has still limitations. Purpose The effects of LCH were investigated on human oral squamous cell carcinoma (OSCC) cells to elucidate its mechanisms. Study Design We explored the mechanism of action of LCH by which it could have effects on JAK2/STAT3 signaling pathway. Methods To confirm LCH anti-cancer effect, analyzed were MTT assay, DAPI staining, soft agar, kinase assay, molecular docking simulation, flow cytometry and Western blotting analysis. Results According to docking and molecular dynamics simulations, the predicted pose of the complex LCH and JAK2 seems reasonable and LCH is strongly bound to active JAK2 with opened activation loop. The LCH inhibitor is surrounded by specific ATP-binding pocket in which it is stabilized by forming hydrogen bonds and hydrophobic interactions. It is shown that LCH plays as a competitive inhibitor in an active state of JAK2. LCH caused a dose-dependent decrease in phosphorylation of JAK2 and STAT3. More interestingly, LCH suppressed JAK2 kinase activity in vitro by its direct binding to the JAK2. LCH significantly inhibited the JAK2/STAT3 signaling pathway, causing the down-regulation of target genes such as Bcl-2, survivin, cyclin D1, p21 and p27. In addition, LCH inhibited cell proliferation and colony formation of OSCC cells in a dose- and time-dependent manner, as well as induction of cell apoptosis through extrinsic and intrinsic pathway. The induction of apoptosis in OSCC cells by LCH was evident in the increased production of ROS, loss of mitochondrial membrane potential, release of cyto c, variation of apoptotic proteins and activation of caspase cascade. Conclusion LCH not only induces apoptosis in OSCC cells through the JAK/STAT3 signaling pathway but also inhibits cell growth. It is proposed that LCH has a promising use for the chemotherapeutic agent of oral cancer.

Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of β-cells, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from α-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3~4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in β-cell replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

Cloned pigs produced by somatic cell nuclear transfer (SCNT) are important as a potential alternative source of organs. Although SCNT has created new possibilities for targeted gene modification, the successful cloning of pigs is rare. Here, we successfully conducted serial SCNT for three generations. We determined that the piglet genome was inherited from donor cell nuclei using microsatellite analysis of each generation. The methylation of differentially methylated regions (DMRs) in H19 was gradually reduced over the three generations of serial SCNT. By contrast, methylation of the insulin-like growth factor 2 (Igf2) DMR increased in the F1 generation, compared to the F0, and remained at the higher level in the F2 and F3 generations. The methylation patterns of housekeeping genes such as GAPDH and β-actin were unchanged in the serially cloned pigs. In addition, expression levels of H19 and Igf2 were variable for each generation of serial SCNT piglets, but there was no clear relationship between the meth...

Most studies of xenografts have been carried out with complex immunosuppressive regimens to prevent immune rejection; however, such treatments may be fatal owing to unknown causes. Here, we performed immune molecular profiling following anti-CD154 monoclonal antibody (mAb) treatment in heterotopic abdominal cardiac xenografts from α-1,3-galactosyltransferase-knockout pigs into cynomolgus monkeys to elucidate the mechanisms mediating the undesirable fatal side effects of immunosuppressive agents. Blood samples were collected from healthy monkeys as control and then at 2 days after xenograft transplantation and just before humane euthanasia; 94 genes related to the immune system were analyzed. The basic immunosuppressive regimen included cobra venom factor, anti-thymocyte globulin, and rituximab, with and without anti-CD154 mAbs. The maintenance therapy was followed with tacrolimus, MMF, and methylprednisolone. The number of upregulated genes was initially decreased on Day 2 (-/+ anti-CD154 mAb, 22/13) and then increased before euthanasia in recipients treated with anti-CD154 mAbs (-/+ anti-CD154 mAb, 30/37). The number of downregulated genes was not affected by anti-CD154 mAb treatment. Additionally, the number of upregulated genes increased over time for both groups. Interestingly, treatment with anti-CD154 mAbs upregulated coagulation inducers (CCL2/IL6) before euthanasia. In conclusion, immunosuppressive regimens used for cardiac xenografting affected upregulation of 6 inflammation genes (CXCL10, MPO, MYD88, NLRP3, TNFα, and TLR1) and downregulation of 8 genes (CCR4, CCR6, CD40, CXCR3, FOXP3, GATA3, STAT4, and TBX21).

BACKGROUND AND PURPOSE Reactive oxygen species (ROS) are potent regulators of stem cell behaviour; however, their physiological significance as regards MMP-mediated regulation of the motility of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) has not been characterized. In the present study, we investigated the role of hydrogen peroxide (H2O2) and associated signalling pathways in promoting UCB-MSCs motility.

Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts.

Pigs are used as potential donor animals for xenotransplantation. However, porcine endogenous retrovirus (PERV), shown to infect both human and non-human primate (NHP) cells in vitro, presents a risk of transmission to humans in xenotransplantation. In this study, we analyzed PERV transmission in various organs after pig-to-NHP xenotransplantation. We utilized pig-to-NHP xenotransplant tissue samples obtained using two types of transgenic pigs from the National Institute of Animal Science (NIAS, Republic of Korea), and examined them for the existence of PERV genes in different organs via PCR and RT-PCR with specific primers. To determine PERV insertion into chromosomes, inverse PCR using PERV long terminal repeat (LTR) region-specific primers was conducted. The PERV gene was not detected in NHP organs in cardiac xenotransplantation but detected in NHP bladders in renal xenotransplantation. The insertion experiment confirmed that PERVs originate from porcine donor cells rather than integrated provirus in the NHP chromosome. We also demonstrate the presence of pig cells in the NHP bladder after renal xenotransplantation using specific-porcine mitochondrial DNA gene PCR. The PERV sequence was detected in the bladder of NHPs after renal xenotransplantation by porcine cell-microchimerism but did not integrate into the NHP chromosome.