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BackgroundExosomes, internal proteins, lipids, and nucleic acids coated by phospholipid bilayer membranes, are one type of small extracellular vesicles, which can mediate cell-cell communication. In recent years, exosomes have gained considerable scientific interest due to their widely applied prospect in the diagnosis and therapeutics of human and animal diseases. In this study, we describe for the first time a feasible method designed to isolate and characterize exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells.ResultsExosomes from feline plasma, urine and adipose-derived mesenchymal stem cells were successfully isolated by differential centrifugation. Quantification and sizing of exosomes were assessed by transmission electron microscopy, flow nano analysis and western blotting. Detected particles showed the normal size (30–100 nm) and morphology described for exosomes, as well as presence of the transmembrane protein (TSG101, CD9, CD63, and CD81) known as exosomal marker.ConclusionsThe results suggest that differential centrifugation is a feasible method for isolation of exosomes from different types of feline samples. Moreover, these exosomes can be used to further diagnosis and therapeutics in veterinary pre-clinical and clinical studies.

Background Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. Results After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. AD-MSCs presented normal karyotype, and did not form in vivo tumors. No adverse events were noted in the case treated with intravenously AD-MSCs. Conclusions Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

Foot-and-mouth disease virus (FMDV) is one of the most important animal pathogens in the world. FMDV naturally infects swine, cattle, and other cloven-hoofed animals. FMD is not adequately controlled by vaccination. An alternative strategy is to develop swine that are genetically resistant to infection. Here, we generated FMDV-specific shRNA transgenic cells targeting either nonstructural protein 2B or polymerase 3D of FMDV. The shRNA-positive transgenic cells displayed significantly lower viral production than that of the control cells after infection with FMDV (P +, CD4+, CD8+, CD21+, and CD172+ cells, and the production of IFN-γ were analyzed, there were no significant differences observed between the TGCS and NS or Non-TGCS, suggesting that the FMDV resistance may be mainly derived from the RNAi-based antiviral pathway. Our work provides a foundation for a breeding approach to preventing infectious disease in swine.

Highlights • We generated transgenic pig expressing PRRSV-specific siRNA. • Stability of siRNA expression was proved in two generations. • Type I interferon was not elicited by the expression of siRNA in vivo. • We proved that transgenic pigs showed substantially decreased virus load in serum after PRRSV infection., Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease causing heavy losses to the swine industry worldwide. Many studies have shown that transient delivery of small interfering RNA (siRNA) or adenovirus-mediated RNA interfere (RNAi) could potentially inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication in vivo and in vitro. Here, we applied RNAi to produce transgenic (TG) pigs that constitutively expressed PRRSV-specific siRNA derived from small hairpin RNA (shRNA). First, we evaluated siRNA expression in the founding and F1 generation pigs and confirmed stable transmission. Then, we detected the expression of IFN-β and protein kinase R (PKR) and found no difference among TG, non-transgenic (NTG), and wild-type pigs. Lastly, the F1 generation pigs, including TG and NTG piglets, were challenged with 3 × 104.5 TCID50 of JXA1, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Our results showed that the in vivo siRNA expression substantially reduced the serum HP-PRRSV titers and increased survival time by 3 days when TG pigs were compared with the NTG controls. These data suggested that RNAi-based genetic modification might be used to breed viral-resistant livestock with stable siRNA expression with no complications of siRNA toxicity.

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time, (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence, (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments, and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34, the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating diseases for the pig industry. Our goal was to identify microRNAs involved in the host immune response to PRRS. We generated microRNA expression profiles of lung tissues from Tongcheng or Landrace pigs infected with a highly pathogenic PRRS virus (PRRSV) at 3, 5, 7 dpi (day post infection) and control individuals from these two breeds. Our data showed that 278 known and 294 novel microRNAs were expressed in these combined microRNA transcriptomes. Compared with control individuals, almost half of the known microRNAs (116 in Tongcheng and 153 in Landrace) showed significantly differential expression (DEmiRNAs) at least once. The numbers of down-regulated DEmiRNAs were larger than the corresponding number of up-regulated DEmiRNAs in both breeds. Interestingly, miR-2320-5p, which was predicted to bind to conserved sequences of the PRRSV genome, was down-regulated significantly at 3 dpi after PRRSV infection in both breeds. In addition, PRRSV infection induced a significant increase of microRNA editing level in both breeds. Our results provide novel insight into the role of microRNA in response to PRRSV infection in vivo, which will aid the research for developing novel therapies against PRRSV.