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Background: Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pigto-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs. Results: We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets. Conclusions: We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.

β-Cell replacement therapy, including allogeneic pancreas and islet transplantation, can normalize HbA1c levels in unstable type 1 diabetic (T1D) patients, but a donor shortage is a serious issue. To overcome this problem, xenotransplantation is an attractive option. In fact, islet transplantation from porcine pancreata was performed in the 1990s, which opened the door for islet xenotransplantation, but the possibility of porcine endogenous retrovirus (PERV) infection was raised, which has restricted progress in this field. The International Xenotransplantation Association published a consensus statement on conditions for undertaking clinical trials of porcine islet products in T1D to restart islet xenotransplantation safely. Clinical porcine islet xenotransplantation was restarted under comprehensive regulations in New Zealand. In addition, newly emerged gene-editing technologies have activated the xenotransplantation field. Islet xenotransplantation is becoming a clinical reality, with the results of recent studies showing promise to advance this field.

To investigate pathogenesis of post-bile duct (BD) injury fibrosis, interlobular BD epithelial injury was induced in male F344 rats by a single IP injection of α-naphthylisothiocyanate (75 mg/kg body weight) and rats were observed for 12 days. On days 1 to 2, cholangiocytes were injured and desquamated. On days 3 to 5, the affected BD began to regenerate, showing positive staining for CK19 and vimentin. On days 5 to 9, fibrotic areas gradually developed around regenerating BD in Glisson's sheath. These consisted of cells positive for vimentin, desmin, and α-SMA; vimentin- and desmin-positive cells were increased in early stage (days 1-3), whereas α-SMA-positive cells appeared in mid (days 4-7) and late stages (days 8-12), although there were cells coexpressing these cytoskeletons. On day 12, BD regeneration almost completed, with reduced fibrosis. Macrophages positive for ED2 (CD163) increased transiently in early stage, whereas those reacting to ED1 (CD68), OX6 (MHC II), and SRA-E5 (CD204) showed a consistent increase throughout the experiment. Interestingly, OX6-positive cells were limited to Glisson's sheath, whereas SRA-E5-positive cells were seen exclusively along sinusoids of hepatic lobules. MCP-1 mRNA increased significantly in early stage. This study shows that macrophages exhibiting different immunophenotypes and distributions participate in post-BD injury fibrosis associated with myofibroblasts expressing various mesenchymal cytoskeletons.

Due to recent advances in research on mesenchymal stem cells (MSCs), MSCs are expected to be used in various clinical applications. However, securing adequate cadaveric donors and safety of living donors are major issues. To solve such issues, we have examined to develop clinical grade neonatal porcine bone marrow‐derived MSCs (npBM‐MSCs). Clinical grade neonatal porcine bone marrow cells were collected, frozen, and sent to our laboratory by air. The npBM‐MSCs were isolated from thawed bone marrow cells, then frozen. The thawed npBM‐MSCs were examined for CD markers and differentiated into chondrocytes, osteocytes, and adipocytes. They were compared with human bone marrow‐derived MSCs (hBM‐MSCs) for growth rate and size. To assess the robustness of proliferation, we compared culture medium with or without gelatin. The npBM‐MSCs expressed positive MSC markers CD29, CD44, and CD90 and were differentiated into chondrocytes, osteocytes, and adipocytes. The doubling time of npBM‐MSCs was significantly shorter than that of hBM‐MSCs (17.3 ± 0.8 vs 62.0 ± 19.6 hours, P < 0.01). The size of npBM‐MSCs was also significantly smaller than that of hBM‐MSCs (13.1 ± 0.3 vs 17.5 ± 0.4 μm, P < 0.001). The npBM‐MSCs showed similar proliferation characters irrespective of with or without gelatin coating. The npBM‐MSCs secreted VEGF‐A, VEGF‐C, and TGF‐β1. We have established npBM‐MSCs which show super‐rapid growth, small size, and robust proliferation profile. The np‐MSCs might be able to solve the donor issues for MSC therapy.

ABSTRACT. Peripheral nerve sheath tumor was found in a 7-year-old male mongrel dog. The tumors were located in the right cheek subcutis and oral submucosa. Histologically, neoplastic cells were arranged in streaming bundles, occasionally interlacing bundles or whorls of elongated and spindle cells. Cellular atypia was poor and mitotic figures were rarely observed. Ultrastructurally, neoplastic cells had basement membrane, typical of Schwann cells. One bundle of normal peripheral nerve fibers and some myelinated axons were seen within the tumor tissues. Immunohistochemically, neoplastic cells reacted to vimentin, glial fibrillary acidic protein, S-100 protein and neuron specific enolase. In addition to the above immunoreactions, the included nerve fibers were positive for myelin basic protein and neurofilament protein. This paper also discusses immunohistochemical findings on differential diagnosis in comparison with those of canine hemangiopericytomas reported hitherto.-KEY WORDS: canine, peripheral nerve sheath tumor (PNST), schwannoma.

Cisplatin (CDDP)-induced renal lesions in rats prove a useful model for analysis of the pathogenesis of post-tubular injury-renal interstitial fibrosis. This study investigated the histopathological changes in 10-day-old neonatal rats induced by a single injection of CDDP (4.5 mg/kg). Compared with age-matched controls, on postinjection (PI) days 1 to 6, the number of apoptotic cells, demonstrable with TUNEL method, was significantly increased in CDDP-treated neonates, and there was no marked epithelial necrosis nor fibrotic lesions. Fibrotic lesions began to be developed solitarily around some nephrons with dilated ducts in the corticomedullary junction on PI day 10 and the lesions became more prominent until PI day 20. The α-SMA-positive myofibroblastic cells were seen exclusively in the fibrotic lesions. Additionally, the numbers of macrophages reacting with ED1 (specific for exudate macrophages), ED2 (for resident macrophages), and OX6 (recognizing MHC class II antigens expressed in antigen-presenting macrophages/dendritic cells) were significantly increased around the affected renal tubules. A greater immunoreaction for TGF-β1 was seen mostly in the renal epithelial cells of CDDP-treated neonates. These findings indicated that macrophage populations and myofibrolastic cells as well as TGF-β1 may be responsible for the production of neonatal renal interstitial fibrosis. Compared with CDDP-injected adult rats that develop extensive interstitial fibrosis (Yamate et al., J Comp Pathol, 1995), the formation of fibrotic lesions was delayed, and the lesions were limited to the area around the affected nephrons; this could be attributable to differences in renal morphology between neonates and mature kidney of adult rats.