The porcine islet-derived organoid showed the characteristics as pancreatic duct.

Organoid is a tissue-engineered organ-like structure that resemble as an organ. Porcine islet-derived organoid might be used as an alternative donor of porcine islet xenotransplantation, a promising therapy for severe diabetes. In this study, we elucidated the characteristics of porcine islet organoids derived from porcine islets as a cell source for transplantation. Isolated porcine islets were 3D-cultured using growth factor-reduced matrigel in organoid culture medium consist of advanced DMEM/F12 with Wnt-3A, R-spondin, EGF, Noggin, IGF-1, bFGF, nicotinamide, B27, and some small molecules. Morphological and functional characteristics of islet organoids were evaluated in comparison with 2D-cultured islets in advanced DMEM/F12 medium. Relatively short-term (approximately 14 days)-cultured porcine islet organoids were enlarged and proliferated, but had an attenuated insulin-releasing function. Long-term (over a month)-cultured islet organoids could be passaged and cryopreserved. However, they showed pancreatic duct characteristics, including cystic induction, strong expression of Sox9, loss of PDX1 expression, and no insulin-releasing function. These findings were seen in long-term-cultured porcine islets. In conclusion, our porcine islet organoids showed the characteristics of pancreatic ducts. Further study is necessary for producing porcine islet-derived organoids having characteristics as islets.

Transplantation of human cells into Interleukin-2 receptor gamma gene knockout pigs under several conditions.

Introduction: Previously, we performed gene knockout (KO) of interleukin-2 receptor gamma (IL2RG) in porcine fetal fibroblasts using zinc finger nuclease-encoding mRNAs, subsequently generating IL2RG KO pigs using these cells through somatic cell nuclear transfer. The IL2RG KO pigs lacked a thymus and were deficient in T lymphocytes and natural killer cells, similar to human X-linked severe combined immunodeficiency (SCID) patients. The present study aimed to evaluate whether pigs can support the growth of xenografted human cells and have the potential to be an effective animal model. Methods: The IL2RG XKOY pigs used in this study were obtained by mating IL2RG XKOX females with wild-type boars. This permitted the routine production of IL2RG KO pigs via natural breeding without complicated somatic cell cloning procedures; therefore, a sufficient number of pigs could be prepared. We transplanted human HeLa S3 cells expressing the tandem dimer tomato into the ears and pancreas of IL2RG KO pigs. Additionally, a newly developed method for the aseptic rearing of SCID pigs was used in case of necessity. Results: Tumors from the transplanted cells quickly developed in all pigs and were verified by histology and immunohistochemistry. We also transplanted these cells into the pancreas of designated pathogen-free pigs housed in novel biocontainment facilities, and large tumors were confirmed. Conclusions: IL2RG KO pigs have the potential to become useful animal models in a variety of translational biology fields.

Generation of heterozygous PKD1 mutant pigs exhibiting early-onset renal cyst formation.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, manifesting as the progressive development of fluid-filled renal cysts. In approximately half of all patients with ADPKD, end-stage renal disease results in decreased renal function. In this study, we used CRISPR-Cas9 and somatic cell cloning to produce pigs with the unique mutation c.152_153insG (PKD1insG/+). Pathological analysis of founder cloned animals and progeny revealed that PKD1insG/+ pigs developed many pathological conditions similar to those of patients with heterozygous mutations in PKD1. Pathological similarities included the formation of macroscopic renal cysts at the neonatal stage, number and cystogenic dynamics of the renal cysts formed, interstitial fibrosis of the renal tissue, and presence of a premature asymptomatic stage. Our findings demonstrate that PKD1insG/+ pigs recapitulate the characteristic symptoms of ADPKD.

Bioresorbable porous ß-tricalcium phosphate chelate-setting cements with poly(lactic-co-glycolic acid) particles as pore-forming agent: fabrication, material properties, cytotoxicity, and in vivo evaluation.

Calcium-phosphate cements (CPCs) have been used as bone filling materials in orthopaedic surgery. However, CPCs are set using an acid-base reaction, and then change into stable hydroxyapatite (HAp) in a living body. Therefore, we developed bioresorbable chelate-setting ß-tricalcium phosphate (ß-TCP) cements based on surface modifications of inositol phosphate (IP6). In order to improve the bioresorbability, we fabricated IP6/ß-TCP cements hybridized with poly(lactic-co-glycolic acid) (PLGA) particles as a pore-forming agent. The compressive strengths of the cements with the amounts of 5 and 10 mass% PLGA particles were 23.2 and 22.8 MPa, respectively. There was no significant difference from cements without PLGA (23.4 MPa). The setting times of the cement specimens with PLGA particles (30 min) were a little longer than those without PLGA particles (26.3 min). The lack of cytotoxicity of the cement specimens was confirmed using osteoblast-like cells (MC3T3-E1). Cylindrical defects were made by drilling into the tibia of mini-pigs and injecting the prepared cement pastes into the defects. Twelve weeks after implantation the specimens were stained with toluidine blue and histologically evaluated. Histological evaluation of cement specimens with PLGA particles showed enhanced bioresorbability. Newly-formed bone was also observed inside cement specimens with PLGA particles. The IP6/ß-TCP cement specimens with PLGA particles had excellent material properties, such as injectability, compressive strength, high porosity, no cytotoxicity in vitro, bioresorption and bone formation abilities in vivo. Organic-inorganic hybridized CPCs are expected to be valuable as novel biodegradable paste-like artificial bone fillers.

Investigation of Clinical Safety of Human iPS Cell-Derived Liver Organoid Transplantation to Infantile Patients in Porcine Model.

Transplantation of liver organoids has been investigated as a treatment alternative to liver transplantation for chronic liver disease. Transportal approach can be considered as a method of delivering organoids to the liver. It is important to set the allowable organoid amount and verify translocation by intraportal transplantation. We first examined the transplantation tolerance and translocation of porcine fetal liver-derived allogeneic organoids using piglets. Fetal liver-derived organoids generated from the Kusabira Orange-transduced pig were transplanted to the 10-day-old piglet liver through the left branch of the portal vein. All recipients survived without any observable adverse events. In contrast, both local and main portal pressures increased transiently during transplantation. In necropsy samples, Kusabira Orange-positive donor cells were detected primarily in the target lobe of the liver and partly in other areas, including the lungs and brain. As we confirmed the transplantation allowance by porcine fetal liver-derived organoids, we performed intraportal transplantation of human-induced pluripotent stem cell (iPSC)-derived liver organoid, which we plan to use in clinical trials, and portal pressure and translocation were investigated. Human iPSC-derived liver organoids were transplanted into the same 10-day-old piglet. Portal hypertension and translocation of human iPSC-derived liver organoids to the lungs were observed in one of two transplanted animals. Translocation occurred in the piglet in which patent ductus venosus (PDV) was observed. Therefore, a 28-day-old piglet capable of surgically ligating PDV was used, and after the PDV was ligated, human iPSC-derived liver organoids with the amount of which is scheduled in clinical trials were transplanted. This procedure inhibited the translocation of human iPSC-derived liver organoids to extrahepatic sites without no portal hypertension. In conclusion, human iPSC-derived liver organoids can be safely transplanted through the portal vein. Ligation of the ductus venosus prior to transplantation was effective in inhibiting extrahepatic translocation in newborns and infants.

Anephrogenic phenotype induced by SALL1 gene knockout in pigs.

To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm of pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and marked dysgenesis of nephron structures were observed in the rudimentary kidney tissue of SALL1-KO foetuses. Biallelic KO mutations of the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 progeny from mutant founder pigs, we identified mutations that could reliably induce the anephrogenic phenotype and hence established a line of fertile SALL1-mutant pigs. Our study lays important technical groundwork for the realization of human kidney regeneration through the use of an empty developmental niche in pig foetuses.

Mycobacterium haemophilum infection with prominent facial manifestation mimicking leprosy.

Mycobacterium haemophilum is a slow-growing non-tuberculous mycobacterium that is rarely known to cause human skin infection, particularly in immunocompromised patients. We recently experienced a 69-year-old Japanese woman with this infection who had been under immunosuppressive treatment for recalcitrant rheumatoid arthritis. The patient showed disseminated erythematous plaques and subcutaneous nodules on the face and extremities, and interestingly, the face manifested with a striking "facies leontina" appearance. Biopsy revealed abscess and granulomatous dermatitis with the involvement of peripheral nerve bundles and the presence of innumerable acid-fast bacilli, thus necessitating differentiation from lepromatous leprosy. M. haemophilum was identified by molecular characterization as well as by successful culture with iron supplements. Although drug susceptibility testing indicated responsiveness to multiple antibiotics administrated simultaneously for the treatment, it took over 6 months to achieve significant improvement, and we also employed concurrent oral potassium iodide administration and repeated surgical excision. This case highlights the importance of continuous combination therapy for successful outcome in this rare infection. Furthermore, application of potassium iodide for mycobacterial infection warrants further evaluation by accumulating more cases.

Generation of naive-like porcine-induced pluripotent stem cells capable of contributing to embryonic and fetal development.

In pluripotent stem cells (PSCs), there are 2 types: naive and primed. Only the naive type has the capacity for producing chimeric offspring. Mouse PSCs are naive, but human PSCs are in the primed state. Previously reported porcine PSCs appear in the primed state. In this study, putative naive porcine-induced pluripotent stem cells (iPSCs) were generated. Porcine embryonic fibroblasts were transduced with retroviral vectors expressing Yamanaka's 4 genes. Emergent colonies were propagated in the presence of porcine leukemia inhibitory factor (pLIF) and forskolin. The cells expressed pluripotency markers and formed embryoid bodies, which gave rise to cell types from all 3 embryonic germ layers. The naive state of the cells was demonstrated by pLIF dependency, 2 active X chromosomes (when female), absent MHC class I expression, and characteristic gene expression profiles. The porcine iPSCs contributed to the in vitro embryonic development (11/24, 45.8%) as assessed by fluorescent markers. They also contributed to the in utero fetal development (11/71, 15.5% at day 23; 1/13, 7.7% at day 65). This is the first demonstration of macroscopic fluorescent chimeras derived from naive-like porcine PSCs, although adult chimeras remain to be produced.