Severe cardiac and skeletal manifestations in DMD-edited microminipigs: an advanced surrogate for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an intractable X-linked muscular dystrophy caused by mutations in the DMD gene. While many animal models have been used to study the disease, translating findings to humans has been challenging. Microminipigs, with their pronounced physiological similarity to humans and notably compact size amongst pig models, could offer a more representative model for human diseases. Here, we accomplished precise DMD modification in microminipigs by co-injecting embryos with Cas9 protein and a single-guide RNA targeting exon 23 of DMD. The DMD-edited microminipigs exhibited pronounced clinical phenotypes, including perturbed locomotion and body-wide skeletal muscle weakness and atrophy, alongside augmented serum creatine kinase levels. Muscle weakness was observed as of one month of age, respiratory and cardiac dysfunctions emerged by the sixth month, and the maximum lifespan was 29.9 months. Histopathological evaluations confirmed dystrophin deficiency and pronounced dystrophic pathology in the skeletal and myocardial tissues, demonstrating that these animals are an unprecedented model for studying human DMD. The model stands as a distinct and crucial tool in biomedical research, offering deep understanding of disease progression and enhancing therapeutic assessments, with potential to influence forthcoming treatment approaches.

Differentiation Capacity of Porcine Skeletal Muscle-Derived Stem Cells as Intermediate Species between Mice and Humans.

Large animal experiments are important for preclinical studies of regenerative stem cell transplantation therapy. Therefore, we investigated the differentiation capacity of pig skeletal muscle-derived stem cells (Sk-MSCs) as an intermediate model between mice and humans for nerve muscle regenerative therapy. Enzymatically extracted cells were obtained from green-fluorescence transgenic micro-mini pigs (GFP-Tg MMP) and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN) fractions. The ability to differentiate into skeletal muscle, peripheral nerve, and vascular cell lineages was examined via in vitro cell culture and in vivo cell transplantation into the damaged tibialis anterior muscle and sciatic nerves of nude mice and rats. Protein and mRNA levels were analyzed using RT-PCR, immunohistochemistry, and immunoelectron microscopy. The myogenic potential, which was tested by Pax7 and MyoD expression and the formation of muscle fibers, was higher in Sk-DN cells than in Sk-34 cells but remained weak in the latter. In contrast, the capacity to differentiate into peripheral nerve and vascular cell lineages was significantly stronger in Sk-34 cells. In particular, Sk-DN cells did not engraft to the damaged nerve, whereas Sk-34 cells showed active engraftment and differentiation into perineurial/endoneurial cells, endothelial cells, and vascular smooth muscle cells, similar to the human case, as previously reported. Therefore, we concluded that Sk-34 and Sk-DN cells in pigs are closer to those in humans than to those in mice.

Sperm Characteristics in Microminipigs.

BACKGROUND/AIM: The microminipig is a relatively new type of mini pig; microminipigs weigh about 10 kg at 6 months of age and are expected to be of use in drug discovery research and safety tests. Herein, we analyzed the characteristics of ejaculated sperm from microminipigs. MATERIALS AND METHODS: Sperm parameters such as microstructure and sensitivity to cold shock were investigated using optical or scanning electron microscopy. RESULTS: Ejaculate volumes and total numbers of sperm were lower than in standard pig strains, but were proportional to body weight. Ejaculation time, pH of the ejaculate, sperm motility and morphology, and sensitivity to cold shock were similar to those of standard pig strains. CONCLUSION: Herein, we provide the first characterization of the ejaculates of microminipigs and demonstrate that this type of pig will be useful not only in medical research, but also in investigations into sperm preservation in different pig breeds.

High Pathological Reproducibility of Diet-induced Atherosclerosis in Microminipigs via Cloning Technology.

BACKGROUND/AIM: The reproducibility of athero - sclerotic lesions was evaluated after the production of cloned-microminipigs and their offspring. MATERIALS AND METHODS: Cloned-microminipig-parents were produced by microminipigsomatic cell nuclei. These parents were crossbred and delivered males (F1-offspring) were divided into two groups: normal chow diet (NcD)-fed and high-fat/high-cholesterol diet (HcD)-fed groups. One of the F1-offsprings was subjected to cloning, and delivered males (F1-clones) were fed with HcD. After 8 weeks, all animals were necropsied for patho - physiological studies compared to non-cloned-microminipigs. RESULTS: HcD-fed F1-offspring and F1-clones, but not NcD-fed F1-offspring, exhibited increased serum lipid levels and systemic atherosclerosis, which were comparable to those of HcD-fed non-cloned-microminipigs. Homogeneity of variance analysis demonstrated that standard deviation values of serum lipoprotein and aortic atherosclerosis area from HcD-fed animals decreased in F1-offspring and F1-clones. CONCLUSION: HcD-induced atherogenesis was highly reproducible in F1-offsprings and F1-clones, indicating that the atherosclerosis-prone genomic background was preserved in the cloned-microminipigs, which can be used for studies on human atherosclerosis and related diseases.

Spontaneous Age-Related Histopathological Changes in Microminipigs.

Microminipigs have become an attractive animal model for toxicology and pharmacology studies and for human disease models, owing to their manageable size. Although there are numerous reports of spontaneous age-related lesions in mice, rats, dogs, and monkeys, those in minipigs are scarce. In the present study, spontaneous age-related histopathological changes were investigated using 37 microminipigs (20 males and 17 females) that were 6 months to 10 years of age. Abnormal deposits of materials were evident in several animals from 6 years of age, and these deposits included amyloid in the renal medulla, thyroid gland, and adrenal gland, hyaline droplets in glomeruli, and fibrillar inclusions in neurons. Arterial sclerosing changes (intimal thickening, intimal proliferation, and medial mineralization) and proliferative lesions (hyperplasia of hepatocytes, follicular cells, Leydig cells, and uterine endometrial glands) were present at 4 years of age and beyond. Renal adenoma, uterine leiomyoma, and Leydig cell tumor were observed in several microminipigs. Moreover, glomerulosclerosis, renal interstitial fibrosis, thymic involution, and adrenocortical cell vacuolation were common in aging microminipigs. Since knowledge of age-related changes is helpful for pathologists, the basic information obtained in this study will be a useful reference for all future toxicity evaluations in microminipigs.

Histological Changes of the Testicular Interstitium during Postnatal Development in Microminipigs.

Microminipigs have become an attractive animal model for the toxicology- and pharmacology-related studies because of their manageable size. In this study, the development of the testicular interstitium and steroidogenesis in microminipigs, from 0 to 12 months of age, were investigated. Testicular interstitium was mostly composed of two types of Leydig cells (large and small Leydig cells) and a few macrophages and mast cells. Large Leydig cells were observed in the peritubular area throughout all the ages. Small Leydig cells were present in the interlobular and subcapsular areas at an early age and then gradually converted into large Leydig cells. Testicular composition of the Leydig cells began to increase after 3 months of age, when spermatogenesis was completed, and reached approximately 35% at 12 months. Steroidogenic enzymes in Leydig cells were detected by immunohistochemistry from 0 month of age. Serum testosterone levels increased substantially from 1.5 to 4.5 months of age, which coincided well with the age of sexual development previously reported in microminipigs. Because the interstitial space of the testis has dramatic variations between species, the basic information obtained in the present study will be a useful reference for all the future toxicity evaluations in microminipigs.

[Current progress of research and use of microminipigs in drug development].

The use of minipigs has been increasing in the areas of pharmacology researches and drug development. The microminipig developed by Fuji Micra Inc. (Shizuoka, Japan) inherits characteristics of other pig strains showing several similarities to humans in anatomy, physiology, omnivorousness and diurnal, but at the same time has several advantages over other pig strains because of its small size which allows easy keeping, handling and dosing, and saving of test substances. The microminipig weighs about 10 kg at the age of 6 months. Canine cages can be used to keep the animal. Swine leukocyte antigens (SLA) are defined in each individual animal which is useful for testing immunological reactions. As there are many similarities in metabolic enzymes and transporters to those in humans, the microminipig is a powerful animal model for toxicokinetic studies. Unfortunately as in other minipigs the microminipig is not appropriate for embryo-fetal development studies of antibody drugs due to its poor placental transfer, but can be used for other reproductive and developmental studies. Repeat dose toxicity, safety pharmacology, immunotoxicity and local tolerance studies should be also other arenas of this animal model.

Reproductive performance and expression of imprinted genes in somatic cell cloned boars.

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin-like growth factor -2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot-and-mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic.

Normal Developmental and Estrous Cycle-dependent Histological Features of the Female Reproductive Organs in Microminipigs.

The microminipig has become an increasingly attractive animal model for various experimental practices because of its manageable size; however, studies of the histological features of the female reproductive organs in microminipigs are limited. The present study investigates the sexual development of the reproductive organs and the cyclical changes during the estrous cycle in female microminipigs. The ovaries, oviducts, uteri, and vaginal tissues from 33 animals aged 0 to 26 months were utilized in this study. By evaluating the large tertiary follicles, corpora lutea, and the regressing corpora lutea, we estimated that female microminipigs reached puberty at approximately 5 months of age and sexual maturity at 8 months of age. The appearance of the follicles and corpora lutea in the ovaries, as well as the epithelium in other reproductive organs, was synchronized with each phase of the estrous cycle and was identical to that in common domestic pigs. In addition, several spontaneous findings were observed, including mesonephric duct remnants adjacent to oviducts and mineralization in ovaries. Understanding the normal histology of the reproductive organs in microminipigs is crucial for advancing pathological evaluations for future toxicological studies.

The Microminipig as an Animal Model for Influenza A Virus Infection.

Pigs are considered a mixing vessel for the generation of novel pandemic influenza A viruses through reassortment because of their susceptibility to both avian and human influenza viruses. However, experiments to understand reassortment in pigs in detail have been limited because experiments with regular-sized pigs are difficult to perform. Miniature pigs have been used as an experimental animal model, but they are still large and require relatively large cages for housing. The microminipig is one of the smallest miniature pigs used for experiments. Introduced in 2010, microminipigs weigh around 10 kg at an early stage of maturity (6 to 7 months old) and are easy to handle. To evaluate the microminipig as an animal model for influenza A virus infection, we compared the receptor distribution of 10-week-old male pigs (Yorkshire Large White) and microminipigs. We found that both animals have SAα2,3Gal and SAα2,6Gal in their respiratory tracts, with similar distributions of both receptor types. We further found that the sensitivity of microminipigs to influenza A viruses was the same as that of larger miniature pigs. Our findings indicate that the microminipig could serve as a novel model animal for influenza A virus infection. IMPORTANCE: The microminipig is one of the smallest miniature pigs in the world and is used as an experimental animal model for life science research. In this study, we evaluated the microminipig as a novel animal model for influenza A virus infection. The distribution of influenza virus receptors in the respiratory tract of the microminipig was similar to that of the pig, and the sensitivity of microminipigs to influenza A viruses was the same as that of miniature pigs. Our findings suggest that microminipigs represent a novel animal model for influenza A virus infection.

Histological Development of Male Reproductive Organs in Microminipigs.

Microminipigs are becoming increasingly attractive alternatives for various experimental applications, such as general toxicology studies, owing to their manageable size. However, there are limited studies on the male reproductive organs of microminipigs, particularly on the histological aspects of sexual maturity. To clarify the development of male reproductive organs, 35 male microminipigs, aged 0 to 12 months, were used in this study. Histological and histomorphological evaluation was performed based on spermatogenic development, measurement of tubular structure in testes and epididymides, and histological progress of accessory glands. In addition, spontaneous testicular changes were quantitatively assessed. Histologically, male microminipigs sexually matured around 4.5 months of age, when spermatogenesis in testes and structural development in genital organs were completed. Spontaneous testicular changes occurred in all the animals investigated. Multinucleated giant cell was most commonly observed, followed by hypospermatogenesis and tubular atrophy/hypoplasia. However, the number of affected tubules was less than 1% in testes after 4.5 months of age, suggesting that the influence of these changes on evaluation of toxicity studies may be minimal. It is preferable to use sexually mature animals in toxicology studies; therefore, the information obtained by the present study will be helpful for future toxicity evaluations in microminipigs.

Preservation and Reproduction of Microminipigs by Cloning Technology.

BACKGROUND/AIM: Microminipigs have been maintained in small populations of closed colonies, involving risks of inbreeding depression and genetic drift. In order to avoid these risks, we assessed the applicability of cloning technology. MATERIALS AND METHODS: Male and female clones were produced from a stock of cryopreserved somatic cells, obtaining offspring by means of natural mating. Phenotypic and genotypic characteristics of original microminipigs, clones and their offspring were analyzed and recorded. RESULTS AND CONCLUSION: Clones presented characteristics similar to those of the cell-stock data. Although the body weight of clones tended to be heavier than that of the cell-stock data, body weights of their offspring were similar to those of previous reports. Thus, cloned microminipigs have the potential to be a valuable genetic resource for reproduction and breeding. Our proposed methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity.

Spermatogenesis in the Microminipig.

The microminipig has considerable potential as an animal model to evaluate general toxicity; however, there are few studies on the male reproductive system, particularly regarding spermatogenesis. The objectives of the present study were to clarify the stages of the seminiferous epithelium cycle on the basis of spermiogenesis and to determine the duration of spermatogenesis in the microminipig. Eleven microminipigs from 6 to 9 months of age were used for histological analyses. Spermiogenesis and stages of the seminiferous epithelium cycle were classified according to the degree of acrosomal development as shown by the periodic acid-Schiff reaction. Three of the animals were intravenously injected with 5-bromo-2-deoxyuridine to determine the duration of spermatogenesis by immunohistochemistry. Spermiogenesis was classified into 15 steps according to the morphological development of the acrosome, nucleus, and flagellum. The seminiferous epithelium cycle was classified into 11 stages based on the steps of spermatid development and germ cell associations. The length of the seminiferous epithelium cycle and the overall spermatogenesis process in the microminipig were estimated to be approximately 9.1 and 40.9 days, respectively. The results indicate the potential application of the microminipig in the evaluation of testicular toxicity, such as spermatogenesis disruption, in general toxicity studies.

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source.

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

Reproductive and growth performance in Jin Hua pigs cloned from somatic cell nuclei and the meat quality of their offspring.

Somatic cell cloning is expected to be a valuable method for conserving genetic resources in pigs. In this study, we compared the reproductive and growth performance of Jin Hua cloned pigs with that of naturally bred Jin Hua pigs. In addition, we generated offspring from the cloned sows and examined the productivity and quality of meat in the progeny. The birth weights and growth rates of somatic cell-cloned pigs were similar to those of Jin Hua pigs. The cloned pigs reached puberty very early, and this is typical of the Jin Hua breed. Furthermore, reproductive performance, in terms of traits such as gestation period, litter size, and raising rate in the cloned pigs were similar to Jin Hua pigs. Although the offspring of the cloned (OC) pigs had lower birth weights than the Jin Hua breed, the daily weight gain of the OC pigs was significantly higher, especially at the finishing stage. The carcass quality of the OC pigs had similar characteristics to the Jin Hua breed, namely thick back fat and a small loin area. Furthermore, the meat qualities of the OC pigs were similar to those of Jin Hua pigs in terms of intramuscular fat content and tenderness. These results demonstrate that cloned pigs and their offspring were similar to the Jin Hua breed in most of the growth, reproductive, and meat productive performances. This strongly suggests that pigs cloned from somatic cell nuclei have the potential to be a valuable genetic resource for breeding.