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The complex process of bone regeneration is influenced by factors such as inflammatory responses, tissue interactions, and progenitor cells. Currently, multiple traumas can interfere with fracture healing, causing the prolonging or failure of healing. In these cases, bone grafting is the most effective treatment. However, there are several drawbacks, such as morbidity at the donor site and availability of suitable materials. Advantages have been provided in this field by a variety of stem cell types. Adipose-derived stem cells (ASCs) show promise. In the radiological examination of this study, it was confirmed that the C/S group showed faster regeneration than the other groups, and Micro-CT also showed that the degree of bone formation in the defect area was highest in the C/S group. Compared to the control group, the change in cortical bone area in the defect area decreased in the sham group (0.874), while it slightly increased in the C/S group (1.027). An increase in relative vascularity indicates a decrease in overall bone density, but a weak depression filled with fibrous tissue was observed outside the compact bone. It was confirmed that newly formed cortical bone showed a slight difference in bone density compared to surrounding normal bone tissue due to increased distribution of cortical bone. In this study, we investigated the effect of bone regeneration by ADMSCs measured by radiation and pathological effects. These data can ultimately be applied to humans with important clinical applications in various bone diseases, regenerative, and early stages of formative differentiation.

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A 25-day-old male common bottlenose dolphin (Tursiops truncatus) died suddenly while swimming at a dolphinarium. The gross examination revealed ulceration on the dorsal and pectoral fins and rostrum. Severe congestion, hemorrhage, and edema were observed in the gastrointestinal tract, liver, mesenteric lymph nodes, lungs, and kidneys. Fibrinosuppurative arthritis of the atlantooccipital joint and extension of fibrin into the spinal canal caused compression of the spinal cord. Histopathological examination revealed tracheitis, fibrinosuppurative bronchopneumonia and enteritis. In the central nervous system, meningeal vessel congestion in the brain, and intraparenchymal hemorrhages with neurodegeneration were observed in the spinal cord. Based on the histopathological findings, representative samples, including lung, liver, mesenteric lymph node, blood obtained from the jugular vein, and fluid sample of the ascites, were inoculated on tryptic soy agar and blood agar for routine bacterial isolation. Each isolated bacterial colony was streaked aseptically onto tryptic soy agar and blood agar for pure culture. After then, polymerase chain reaction (PCR) was performed for further identification of pathogenic microorganisms. PCR identified Escherichia fergusonii, Shewanella haliotis, Enterococcus faecalis, and Staphylococcus schleiferi. E. fergusonii was considered the primary etiologic agent in this case since it was the only species identified in all representative samples. The cause of death in this animal was E. fergusonii sepsis. To the best of our knowledge, this is the first case of neonatal sepsis associated with E. fergusonii infection in a dolphin, and suggests E. fergusonii as an opportunistic pathogen associated with sepsis in dolphins.

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Myxoid pleomorphic liposarcoma is characterized by pathological features of both pleomorphic liposarcoma and myxoid liposarcoma, as the name suggests. In this case, a myxoid pleomorphic liposarcoma was observed in a 5-year-old male African pygmy hedgehog. It consisted of ~60% of the myxoid substance area with proliferating round cells and ~30% of pleomorphic neoplastic cells. The subject presented with extrapulmonary metastasis, but a good prognosis during 6 months of follow-up, which is similar to the characteristics of myxoid liposarcoma. The histopathological features of myxoid pleomorphic liposarcoma may reflect the features of either myxoid liposarcoma or pleomorphic liposarcoma depending on the proportion of each histopathological feature. The proportion of the pleomorphic area and the myxoid area may offer information on the prognosis and metastasis of myxoid pleomorphic liposarcoma, which will be helpful for setting up a treatment plan. Thus, analyzing the proportion of pleomorphic area and myxoid area could be suggested as one of the ways to predict clinical outcomes. In addition to the fact that this is the first case of a myxoid pleomorphic liposarcoma in hedgehogs, this case is meaningful, considering the unique histopathological characteristics and rare incidence of myxoid pleomorphic liposarcoma that could be important in humans as well.

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BACKGROUND: Although there are growing demands for stem cell-based therapy for companion animals in various diseases, a few clinical trials have been reported. Moreover, most of them are the results from only one or a few times of stem cell injection. OBJECTIVES: The aim of this study is to describe a long-term treatment with allogeneic adipose-derived stem cells (ASCs) in a dog with rheumatoid arthritis (RA), which is a rare canine disease. METHODS: The dog with RA received intravascular injection of allogeneic ASCs derived from two healthy donors once a month for 11 months. To assess therapeutic effects of ASCs, orthopedic examination and clinical evaluation was performed. Cytokines of tumor necrosis factor-α and interleukin-6 in the plasma were measured using ELISA analysis. RESULTS: Despite this repeated and long-term administration of allogeneic ASCs, there were no side effects such as immunorejection responses or cell toxicity. The orthopedic examination score for the dog decreased after ASCs treatment, and the clinical condition of the dog and owner's satisfaction were very good. CONCLUSIONS: Although ASCs has been suggested as one of the options for RA treatment because of its anti-inflammatory and immunosuppressive functions, it has never been used to treat RA in dogs. The present report describes a case of canine RA treated with allogeneic ASCs for long-term in which the dog showed clinical improvement without adverse effects.

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Sirt2 regulates various biological processes by deacetylating target genes. Despite roles in regulating proliferation, cell cycle, and glucose metabolism, which are closely associated with skeletal muscle physiology, Sirt2 functions in this tissue remain unclear. In this study, genetic deletion of Sirt2 delayed muscle regeneration after Notexin-induced muscle injury. Gene expressions of myogenic regulatory factors, including Myf5, MyoD, and Myogenin, and cell cycle regulators, such as cyclin D1 and CDK2, were repressed in Sirt2 knockout mice after injury. Also, Sirt2 knockout mice presented muscle atrophy after muscle injury which is associated with the down-regulation of anabolic signaling and the up-regulation of catabolic signaling, in particular, increased atrogin1 transcriptional expression. Thus, Sirt2 positively regulated skeletal muscle regeneration after muscle injury by regulating transcriptional expression involved in myogenesis, cell cycle, and anabolic and catabolic signaling. Based on the in vivo analyses, Sirt2 could function as an interventional therapeutic for chronic myopathy, which is characterized by impaired muscle regeneration and muscle atrophy.

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AIMS: Alcoholic liver disease (ALD) comprises an important component in chronic liver diseases, and its clinical significance has increased due to the high consumption of alcohol worldwide. Vitamin C is a potent antioxidant, and several previous studies have suggested that its therapeutic role in ALD is derived from its antioxidant role. However, its anti-inflammatory role in ALD remains to be elucidated. Especially, the relationship between vitamin C and infiltration of neutrophils in ALD has not been discussed to date. For the reason, the present study investigated the precise role of vitamin C in neutrophil infiltration in ALD. MAIN METHODS: In the present study, wild-type C57BL/6 and vitamin C-deficient senescence marker protein 30-knockout mice were pair-fed with a Lieber-DeCarli control or ethanol diet. Ethanol-fed groups were fed with increasing concentrations of EtOH (Lieber-DeCarli control diet for 5 days, 3% EtOH diet for a week, and 5% diet for 2 weeks) with or without vitamin C supplementation. KEY FINDINGS: Vitamin C dramatically attenuated the ethanol-mediated liver disease in the vitamin C-deficient ethanol-fed mice group by suppressing the infiltration of neutrophils accompanied by less CD68-positive cell infiltration. This attenuating role of vitamin C in neutrophil infiltration in the liver is associated with its protective effect for the ethanol-mediated intestinal damage in vitamin C-deficient ethanol-fed mice. SIGNIFICANCE: This study provides a novel possibility of vitamin C to be used as an anti-inflammatory therapeutic agent associated with neutrophil infiltration in ALD, thereby helping to establish strategies for attenuating ALD.

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AIM: Sirtuin 2 (SIRT2) is a NAD+-dependent deacetylase involved in various biological functions via deacetylation of proteins, including histone protein. Hepatic fat accumulation from aging and excess caloric intake contribute to development of non-alcoholic fatty liver disease. The study aim was to elucidate the role of SIRT2 in lipid metabolism homeostasis. MATERIALS AND METHODS: SIRT2+/+ (C57BL/6) and SIRT2-/- were randomly assigned to normal diet or high-fat diet (HFD) groups and fed for 6 weeks. Histological features of the livers were evaluated by hematoxylin and eosin and Masson's trichrome staining, and the levels of selected factors were determined by quantitative reverse transcription-polymerase chain reaction and western blot analysis. KEY FINDINGS: Although the SIRT2-/- mice were viable, their livers exhibited higher glycogen accumulation, and skeletal muscle showed features of increased metabolic demand. The SIRT2-/- mice attenuated HFD-induced weight gain, visceral adipose tissue formation, and fat accumulation in the liver in which the expressions of genes involved in metabolic substrate transport were modified. Additionally, the hepatocellular senescence and upregulated cell-cycle factors upon HFD intake in SIRT2-/- livers suggested a role of SIRT2 in gene expression during abnormal metabolism. Moreover, the fibrotic phenotype of liver tissue without fat accumulation and the increased expression of genes involved in liver fibrosis in the HFD-fed SIRT2-/- mice indicated that SIRT2 had a role in hepatocyte and hepatic stellate cell activation. SIGNIFICANCE: Our results indicated that SIRT2 has a critical role in regulating lipid metabolic homeostasis and in sustaining liver integrity by modulating related gene expression.

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Despite the increasing clinical importance of nonalcoholic fatty liver disease (NAFLD), little is known about its underlying pathogenesis or specific treatment. The senescence marker protein 30 (SMP30), which regulates the biosynthesis of vitamin C (VC) in many mammals, except primates and humans, was recently recognized as a gluconolactonase. However, the precise relation between VC and lipid metabolism in NAFLD is not completely understood. Therefore, this study aimed to clearly reveal the role of VC in NAFLD progression. SMP30 knockout (KO) mice were used as a VC-deficient mouse model. To investigate the precise role of VC on lipid metabolism, 13- to 15-week-old SMP30 KO mice and wild-type mice fed a 60% high-fat diet were exposed to tap water or VC-containing water (1.5 g/L) ad libitum for 11 weeks. Primary mouse hepatocytes isolated from the SMP30 KO and wild-type mice were used to demonstrate the relation between VC and lipid metabolism in hepatocytes. Long-term VC deficiency significantly suppressed the progression of simple steatosis. The high-fat diet-fed VC-deficient SMP30 KO mice exhibited impaired sterol regulatory element-binding protein-1c activation because of excessive cholesterol accumulation in hepatocytes. Long-term VC deficiency inhibits de novo lipogenesis through impaired sterol regulatory element-binding protein-1c activation.

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Hepatic fibrosis is a common liver disease caused by excessive collagen deposition in the liver. Since liver transplantation is the only current treatment for cirrhosis with worsened fibrosis, a new strategy to develop anti-fibrosis drugs with no adverse effects is necessary. In recent studies, amino acids have been applied as a type of therapy in various fields. l-serine plays a major role in antioxidant production via the maintenance of nicotinamide adenine dinucleotide phosphate hydride production in the mitochondria. l-serine may reduce fibrotic lesions in a mouse model of chronic liver injury. This study used 27 six-week-old C57BL/6 mice and injected them three times a week for eight weeks with carbon tetrachloride (CCl4) (1.5 mg/kg, 10% v/v CCl4 in olive oil) to create a hepatic fibrosis mouse model. The mice, which weighed approximately 20-30 g, were randomly classified into four groups: 1) the olive oil group, which received intraperitoneal injection of olive oil (1.5 mg/kg, 3 times per week for 8 weeks); 2) the CCl4-only group; 3) the CCl4 + losartan (10 mg/kg, PO, 5 days on, weekend off for 8 weeks) group; and 4) the CCl4 + l-serine (100 g/L, free access for 8 weeks) group. Hematoxylin and eosin staining and Masson's trichrome staining showed reduced inflammatory cell deposition and collagen deposition in the liver tissue in the l-serine supplemented group. l-serine was found to reduce the spread of hepatic fibrosis and has potential use in clinical settings. Based on these histopathological observations, l-serine is a potential anti-fibrosis drug.

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BACKGROUND/AIM: The pathological role of vascular endothelial growth factor receptor 2 (VEGFR-2) in chronic liver injury and liver regeneration is not fully understood. This study analysed the role of VEGFR-2 in liver fibrosis and its regeneration process. MATERIALS AND METHODS: We administered intraperitoneally 50 mg/kg to 300 mg/kg thioacetamide (TAA) to 9-week-old male mice for 17 weeks. We measured levels of VEGFR-2 protein and identified the location of cells that specifically express VEGFR-2. RESULTS: VEGFR-2 is rarely expressed in normal hepatocytes. However, high VEGFR-2 expression in liver sinusoidal endothelial cells was noted in the TAA group. Conversely, the group that experienced regeneration from liver fibrosis showed significantly higher VEGFR-2 expression in the nucleus of hepatocytes compared to the other groups. CONCLUSION: VEGFR-2 plays a pivotal role in the nucleus of hepatocytes during liver regeneration and VEGFR-2 may be closely related to cell division. Therefore, VEGFR-2 may be a new therapeutic target for liver regeneration.

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Senescence marker protein 30 (SMP30) is a cell survival factor playing an important role in vitamin C synthesis and antiapoptosis. Moreover, its cytoprotective role suggests a possibility to be related to cancer cell survival. Mammary carcinoma is a common cancer in both humans and animals. Because of its histopathological diversity, especially in the early stage, histopathological diagnosis may be complicated; therefore, a diagnostic marker is helpful for confirmation. The present study analyzed the expression pattern of SMP30 in mammary carcinoma in humans, dogs, and cats. Immunohistochemistry, immunofluorescence, and western blot analysis were used to investigate SMP30 expression patterns. The expression was specifically observed in neoplastic glandular epithelial cells. The expression increased with the malignancy of glandular epithelial cells with a highly proliferative status. However, SMP30 expression was low in normal mammary gland tissues or well-differentiated adenoma tissues. The patterns were consistently reproduced in canine primary mammary carcinoma cells and MCF-7 and MDA-MB-231 human carcinoma cell lines. This study provides useful information to understand SMP30 expression in various stages of mammary carcinoma and to suggest its utility as a pan-species diagnostic marker, thereby helping to establish strategies for diagnosing mammary carcinoma in several species.

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Nogo-A (Rtn 4A), a member of the reticulon 4 (Rtn4) protein family, is a neurite outgrowth inhibitor protein that is primarily expressed in the central nervous system (CNS). However, previous studies revealed that Nogo-A was upregulated in skeletal muscles of Amyotrophic lateral sclerosis (ALS) patients. Additionally, experiments showed that endoplasmic reticulum (ER) stress marker, C/EBP homologous protein (CHOP), was upregulated in gastrocnemius muscle of a murine model of ALS. We therefore hypothesized that Nogo-A might relate to skeletal muscle diseases. According to our knocking down and overexpression results in muscle cell line (C2C12), we have found that upregulation of Nogo-A resulted in upregulation of CHOP, pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, while downregulation of Nogo-A led to downregulation of CHOP, IL-6 and TNF-α. Immunofluorescence results showed that Nogo-A and CHOP were expressed by myofibers as well as tissue macrophages. Since resident macrophages share similar functions as bone marrow-derived macrophages (BMDM), we therefore, isolated macrophages from bone marrow to study the role of Nogo-A in activation of these cells. Lipopolysaccharide (LPS)-stimulated BMDM in Nogo-KO mice showed low mRNA expression of CHOP, IL-6 and TNF-α compared to BMDM in wild type (WT) mice. Interestingly, Nogo knockout (KO) BMDM exhibited lower migratory activity and phagocytic ability compared with WT BMDM after LPS treatment. In addition, mice experiments data revealed that upregulation of Nogo-A in notexin- and tunicamycin-treated muscles was associated with upregulation of CHOP, IL-6 and TNF-α in WT group, while in Nogo-KO group resulted in low expression level of CHOP, IL-6 and TNF-α. Furthermore, upregulation of Nogo-A in dystrophin-deficient (mdx) murine model, myopathy and Duchenne muscle dystrophy (DMD) clinical biopsies was associated with upregulation of CHOP, IL-6 and TNF-α. To the best of our knowledge, this is the first study to demonstrate Nogo-A as a regulator of inflammation in diseased muscle and bone marrow macrophages and that deletion of Nogo-A alleviates muscle inflammation and it can be utilized as a therapeutic target for improving muscle diseases.

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Among the three isoforms encoded by Rtn4, Nogo-A has been intensely investigated as a central nervous system inhibitor. Although Nogo-A expression is increased in muscles of patients with amyotrophic lateral sclerosis, its role in muscle homeostasis and regeneration is not well elucidated. In this study, we discovered a significant increase in Nogo-A expression in various muscle-related pathological conditions. Nogo-/- mice displayed dystrophic muscle structure, dysregulated muscle regeneration following injury, and altered gene expression involving lipid storage and muscle cell differentiation. We hypothesized that increased Nogo-A levels might regulate muscle regeneration. Differentiating myoblasts exhibited Nogo-A upregulation and silencing Nogo-A abrogated myoblast differentiation. Nogo-A interacted with filamin-C, suggesting a role for Nogo-A in cytoskeletal arrangement during myogenesis. In conclusion, Nogo-A maintains muscle homeostasis and integrity, and pathologically altered Nogo-A expression mediates muscle regeneration, suggesting Nogo-A as a novel target for the treatment of myopathies in clinical settings.

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The therapeutic effects of mesenchymal stem cells (MSCs) in musculoskeletal diseases (MSDs) have been verified in many human and animal studies. Although some tissues contain MSCs, the number of cells harvested from those tissues and rate of proliferation in vitro are not enough for continuous transplantation. In order to produce and maintain stable MSCs, many attempts are made to induce differentiation from pluripotent stem cells (iPSCs) into MSCs. In particular, it is also known that the paracrine action of stem cell-secreted factors could promote the regeneration and differentiation of target cells in damaged tissue. MicroRNAs (miRNAs), one of the secreted factors, are small non-coding RNAs that regulate the translation of a gene. It is known that miRNAs help communication between stem cells and their surrounding niches through exosomes to regulate the proliferation and differentiation of stem cells. While studies have so far been underway targeting therapeutic miRNAs of MSDs, studies on specific miRNAs secreted from MSCs are still minimal. Hence, our ultimate goal is to obtain sufficient amounts of exosomes from iPSC-MSCs and develop them into therapeutic agents, furthermore to select specific miRNAs and provide safe cell-free clinical setting as a cell-free status with purpose of delivering them to target cells. This review article focuses on stem cell therapy on MSDs, specific microRNAs regulating MSDs and updates on novel approaches.

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Myofibromas are mesenchymal tumours of myofibroblastic origin that occur in solitary or multicentric forms. Solitary benign myofibromas mainly occur on the head and neck, especially in the subcutaneous region. They rarely occur in visceral organs in humans, but visceral myofibroma has not been reported in animals. We now report a case of testicular myofibroma in a 6-year-old rabbit in which orchiectomy revealed an enlarged testis with a multinodular surface. The cut surface of the testis showed a thick, homogeneous white-yellow mass surrounding the testicular parenchyma. Histopathologically, the mass was composed of collagen and eosinophilic fascicles of spindle cells that were immunopositive for α-smooth muscle actin but not desmin, S-100 or von Willebrand factor. These features distinguished the myofibroma from other spindle cell tumours. To the best of our knowledge, this is the first report of solitary testicular myofibroma in any animal species.

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It is hard to supply satellite cells as a cell source for therapy of muscle degenerative disease since the sampling of muscle tissue is very invasive to a patient with muscular disease. Direct conversion allows us to get specific cell types by transduction of defined transcriptional factors. To induce myogenic direct conversion, we transduced five transcriptional factors including Pax3, Sox2, Klf4, c-Myc, and Esrrb into mouse embryonic fibroblasts. We found that the transduction of the five transcriptional factors induced myogenic direct conversion of fibroblast. We revealed that the transduced cells with the five transcriptional factors were converted to myogenic lineage cells through a paraxial mesoderm-like stage. The expression level of myogenic-related genes of the transduced cells gradually increased as the passage increased. The induced myogenic lineage cells differentiated into muscle fibers in virto and in vivo. The current study revealed that the five transcription factors generated myogenic lineage cells from fibroblast via a paraxial mesoderm stage. The induced myogenic lineage cells may have a potential being applied as cell source for degenerative muscle disease.

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Tumor incidence in wild mammals is reportedly very low. Wild nutria, a large rodent, is known to carry many infectious diseases, but rarely exhibits neoplastic diseases. We necropsied a male wild nutria and found a large nodular mass in the left inguinal region, adjacent to the penis. Histopathologically, the mass was diagnosed as preputial gland adenoma. Spontaneous preputial gland adenomas are extremely rare in all animals. Moreover, reports of tumors in nutrias have been limited to adenocarcinomas of the lungs and uterus, as well as subcutaneous fibromas. Here, we describe preputial gland adenoma in a wild nutria.

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Gene therapy is emerging as an effective treatment option for various inherited genetic diseases. Gutless adenovirus (GLAd), also known as helper-dependent adenovirus (HDAd), has many notable characteristics as a gene delivery vector for this particular type of gene therapy, including broad tropism, high infectivity, a large transgene cargo capacity, and an absence of integration into the host genome. Additionally, GLAd ensures long-term transgene expression in host organisms owing to its minimal immunogenicity, since it was constructed following the deletion of all the genes from an adenovirus. However, the clinical use of GLAd for the treatment of inherited genetic diseases has been hampered by unavoidable contamination of the highly immunogenic adenovirus used as a helper for GLAd production. Here, we report the production of GLAd in the absence of a helper adenovirus, which was achieved with a helper plasmid instead. Utilizing this helper plasmid, we successfully produced large quantities of recombinant GLAd. Importantly, our helper plasmid-based system exclusively produced recombinant GLAd with no generation of helper plasmid-originating adenovirus and replication-competent adenovirus (RCA). The recombinant GLAd that was produced efficiently delivered transgenes regardless of their size and exhibited therapeutic potential for Huntington's disease (HD) and Duchenne muscular dystrophy (DMD). Our data indicate that our helper plasmid-based GLAd production system could become a new platform for GLAd-based gene therapy.

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In previous work, we established an equine induced pluripotent stem cell line (E-iPSCs) from equine adipose-derived stem cells (ASCs) using a lentiviral vector encoding four transcription factors: Oct4, Sox2, Klf4, and c-Myc. In the current study, we attempted to differentiate these established E-iPSCs into mesenchymal stem cells (MSCs) by serial passaging using MSC-defined media for stem cell expansion. Differentiation of the MSCs was confirmed by analyzing expression levels of the MSC surface markers CD44 and CD29, and the pluripotency markers Nanog and Oct4. Results indicated that the E-iPSC-derived MSCs (E-iPSC-MSCs) retained the characteristics of MSCs, including the ability to differentiate into chondrogenic, osteogenic, or myogenic lineages. E-iPSC-MSCs were rendered suitable for therapeutic use by inhibiting immune rejection through exposure to transforming growth factor beta 2 (TGF-ß2) in culture, which down-regulated the expression of major histocompatibility complex class I (MHC class I) proteins that cause immune rejection if they are incompatible with the MHC antigen of the recipient. We reported 16 cases of E-iPSC-MSC transplantations into injured horses with generally positive effects, such as reduced lameness and fraction lines. Our findings indicate that E-iPSC-MSCs can demonstrate MSC characteristics and be safely and practically used in the treatment of musculoskeletal injuries in horses.

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A black bear of 29-year-old (Ursus americanus) died unexpectedly in captivity without any gross lesions or clinical signs. We identified a firm, lobulated, yellowish tan, and well-circumscribed mass embedded inside the testicular tissue at the time of necropsy. The tumor sections exhibited soft necrotic and hemorrhagic areas beneath its capsule. Histologically, the tumor comprised Sertoli cells arranged in tubules and solid sheets supported by prominent fibrous connective tissues. The Sertoli cells were positive for vimentin and ER-ß expression, whereas it showed negative staining for inhibin-α, cytokeratin 19, and S-100. To the best of our knowledge, this is the rare case report of testicular Sertoli cell tumor in black bear.