A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

Characteristics of the Dental Pulp and Periodontal Ligament Stem Cells of the Yucatan Miniature Pig

Miniature pigs have been considered as a recommended large animal model for biomedical research. Mesenchymal stem cells offer promising potential for tissue regeneration. Recent studies have suggested that dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) may provide more reliable strategies for the treatment of dental diseases using a cell-based tissue engineering approach. The aim of this study was to isolate and compare the characteristics of the DPSCs and PDLSCs of a miniature pig breed to the DPSCs and PDLSCs of a domestic farm pig breed. Stem cells of the DP and PDL were obtained from a male Yucatan miniature pig (nine months old) and a male domestic farm pig breed (six months old). The cell morphology, surface stem cell marker expression, proliferation, and osteogenic differentiation ability were evaluated. Under a light microscope, the DPSCs and PDLSCs of the miniature pig breed had morphologies similar to those of the domestic farm pig breed. The proliferation of PDLSCs in both animals showed no significant differences, except on day five, whereas the proliferation of DPSCs was significantly higher in the miniature pig breed. However, the osteogenic abilities of the DPSCs and PDLSCs from the miniature pig breed were significantly lower compared to the domestic farm pig breed. This observation emphasizes the need for the breed-specific optimization of an osteogenic differentiation culture protocol for Yucatan miniature pig DPSCs and PDLSCs before application to cell-based therapy for tissue engineering and regenerative medicine.

Establishment of Acute Liver Failure Model in Tibet Miniature Pig and Verified by a Non-bioartificial Liver

Background and aims Acute liver failure (ALF) is a severe liver disease with high morbidity and mortality. Animal model is very important to research ALF. This study aimed to establish a reproducible, D-galactosamine-induced, Tibet miniature pig model of ALF and verified by a non-bioartificial liver (NBAL).Methods Thirteen Tibet miniature pigs were randomly divided into four groups (A, B, C, D) after central venous catheterization, then D-galactosamine (D-gal) at 0.45, 0.40 and 0.35g/kg body weight were injected through the central venous catheter in group A, B, C. While in group D, D-gal at 0.35g/kg body weight was injected and treated by a NBAL at 48 h after D-gal administration. Vital signs, blood index values were recorded at every 12 h after D-gal administration and every 2 h during NBAL treatemnt. Meanwhile, clinical manifestations, survival times and results of H&E staining, Tunel, Ki67, Masson assays and Picrosirius red staining were performed.Results Tibet miniature pigs developed different degrees of debilitation, loss of appetite and jaundice after D-gal administration. Survival times of groups A, B, C and D were 39.7±5.9 h, 53.0±12.5 h, 61.3±8.1 h and 61±7 h, respectively. Blood levels of ALT, AST, TBIL, Cr, BUN, Amm, PT and inflammation factors (such as TNF-α, IL-1β, IL-6) levels significantly increased compared with baseline in different groups (Ps < 0.05). Pathological results showed obvious liver cell necrosis positively correlated to the dose of D-gal. However, the NBAL did not increase the survival times of ALF, neither Amm and liver cell necrosis, it just decreased some biochemistry indexes (such as TBIL, ALT, AST).Conclusions: We successfully established a reproducible, D-galactosamine-induced, Tibet miniature pig model of ALF, the NBAL did not improve the survival times of ALF and we think the dosage of 0.35 g/kg is optimal.

Spatiotemporal Expression Patterns of Critical Genes Involved in FGF Signaling during Morphogenesis and Odontogenesis of Deciduous Molar in Miniature Pig

Background The fibroblast growth factor (FGF) pathway plays important role in epithelial-mesenchymal interactions during tooth development. However, how the ligands, receptors, and inhibitors of the FGF pathway get involved into the epithelial-mesenchymal interactions are largely unknown in miniature pigs, which can be used as large animal models for similar tooth anatomy and replacement patterns to humans. Results In this study, we investigated the spatiotemporal expression patterns of critical genes encoding FGF ligands, receptors, and inhibitors in the third deciduous molar of the miniature pig at the cap, early bell, and late bell stages. With the methods of fluorescence in situ hybridization and real time RT-PCR, it was revealed that the expression of Fgf3, Fgf4, Fgf7, and Fgf9 mRNAs were located mainly in the dental epithelium and underlying mesenchyme at the cap stage. The expression levels of Fgf3 and Fgf7 in the mesenchyme were upregulated in the early bell stage and then concentrated in the odontoblasts layer in the late bell stage. In contrast, the expression levels of Fgf4 and Fgf9 in the mesenchyme were downregulated from the cap to bell stage. Gene expression analysis also suggested that Fgfr1 and Fgfr3 were the major receptors regulating dental calcification. Furthermore, the inhibitor-coding genes Sprouty 2 and Sprouty 4 were expressed in the epithelium and mesenchyme in the three stages, indicating that elaborate regulation occurred during dental morphogenesis. Conclusions The spatiotemporal expression pattern of FGF signaling provides the foundation for future studies aiming to fine-tune dental morphogenesis and odontogenesis by controlling the interactions between the dental epithelium and mesenchyme, thereby promoting tooth regeneration in large mammals.

Prevention of Chronic Rejection of Marginal Kidney Graft by Using a Hydrogen Gas-Containing Preservation Solution and Adequate Immunosuppression in a Miniature Pig Model

In clinical kidney transplantation, the marginal kidney donors are known to develop chronic allograft rejection more frequently than living kidney donors. In our previous study, we have reported that the hydrogen gas-containing organ preservation solution prevented the development of acute injuries in the kidney of the donor after cardiac death by using preclinical miniature pig model. In the present study, we verified the impact of hydrogen gas treatment in transplantation with the optimal immunosuppressive protocol based on human clinical setting by using the miniature pig model. Marginal kidney processed by hydrogen gas-containing preservation solution has been engrafted for long-term (longer than 100 days). A few cases showed chronic rejection reaction; however, most were found to be free of chronic rejection such as graft tissue fibrosis or renal vasculitis. We concluded that marginal kidney graft from donor after cardiac death is an acceptable model for chronic rejection and that if the transplantation is carried out using a strict immunosuppressive protocol, chronic rejection may be alleviated even with the marginal kidney.