Material Design of Porous Hydroxyapatite Ceramics via Inverse Analysis of an Estimation Model for Bone-Forming Ability Based on Machine Learning and Experimental Validation of Biological Hard Tissue Responses.

Hydroxyapatite and ß-tricalcium phosphate have been clinically applied as artificial bone materials due to their high biocompatibility. The development of artificial bones requires the verification of safety and efficacy through animal experiments; however, from the viewpoint of animal welfare, it is necessary to reduce the number of animal experiments. In this study, we utilized machine learning to construct a model that estimates the bone-forming ability of bioceramics from material fabrication conditions, material properties, and in vivo experimental conditions. We succeeded in constructing two models: 'Model 1', which predicts material properties from their fabrication conditions, and 'Model 2', which predicts the bone-formation rate from material properties and in vivo experimental conditions. The inclusion of full width at half maximum (FWHM) in the feature of Model 2 showed an improvement in accuracy. Furthermore, the results of the feature importance showed that the FWHMs were the most important. By an inverse analysis of the two models, we proposed candidates for material fabrication conditions to achieve target values of the bone-formation rate. Under the proposed conditions, the material properties of the fabricated material were consistent with the estimated material properties. Furthermore, a comparison between bone-formation rates after 12 weeks of implantation in the porcine tibia and the estimated bone-formation rate. This result showed that the actual bone-formation rates existed within the error range of the estimated bone-formation rates, indicating that machine learning consistently predicts the results of animal experiments using material fabrication conditions. We believe that these findings will lead to the establishment of alternative animal experiments to replace animal experiments in the development of artificial bones.

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.

Development of a panel for detection of pathogens in xenotransplantation donor pigs.

There have been high expectations in recent years of using xenotransplantation and regenerative medicine to treat humans, and pigs have been utilized as the donor model. Pigs used for these clinical applications must be microbiologically safe, that is, free of infectious pathogens, to prevent infections not only in livestock, but also in humans. Currently, however, the full spectrum of pathogens that can infect to the human host or cause disease in transplanted porcine organs/cells has not been fully defined. In the present study, we thus aimed to develop a larger panel for the detection of pathogens that could potentially infect xenotransplantation donor pigs. Our newly developed panel, which consisted of 76 highly sensitive PCR detection assays, was able to detect 41 viruses, 1 protozoa, and a broad range of bacteria (by use of universal 16S rRNA primers). The applicability of this panel was validated using blood samples from uterectomy-born piglets, and pathogens suspected to be vertically transmitted from sows to piglets were successfully detected. We estimate that, at least for viruses and bacteria, the number of target pathogens detected by the developed screening panel should suffice to meet the microbiological safety levels required worldwide for xenotransplantation and/or regenerative therapy. This panel provides greater diagnosis options to produce donor pigs so that it would render unnecessary to screen for all pathogens listed. Instead, the new panel could be utilized to detect only required pathogens within a given geographic range where the donor pigs for xenotransplantation have been and/or are being developed.

Phenotypic features of genetically modified DMD-XKOXWT pigs.

Introduction: Duchenne muscular dystrophy (DMD) is a hereditary neuromuscular disorder caused by mutation in the dystrophin gene (DMD) on the X chromosome. Female DMD carriers occasionally exhibit symptoms such as muscle weakness and heart failure. Here, we investigated the characteristics and representativeness of female DMD carrier (DMD-XKOXWT) pigs as a suitable disease model. Methods: In vitro fertilization using sperm from a DMD-XKOY↔XWTXWT chimeric boar yielded DMD-XKOXWT females, which were used to generate F2 and F3 progeny, including DMD-XKOXWT females. F1-F3 piglets were genotyped and subjected to biochemical analysis for blood creatine kinase (CK), aspartate aminotransferase, and lactate dehydrogenase. Skeletal muscle and myocardial tissue were analyzed for the expression of dystrophin and utrophin, as well as for lymphocyte and macrophage infiltration. Results: DMD-XKOXWT pigs exhibited various characteristics common to human DMD carrier patients, namely, asymptomatic hyperCKemia, dystrophin expression patterns in the skeletal and cardiac muscles, histopathological features of skeletal muscle degeneration, myocardial lesions in adulthood, and sporadic death. Pathological abnormalities observed in the skeletal muscles in DMD-XKOXWT pigs point to a frequent incidence of pathological abnormalities in the musculoskeletal tissues of latent DMD carriers. Our findings suggest a higher risk of myocardial abnormalities in DMD carrier women than previously believed. Conclusions: We demonstrated that DMD-XKOXWT pigs could serve as a suitable large animal model for understanding the pathogenic mechanism in DMD carriers and developing therapies for female DMD carriers.

Phenotypic features of dystrophin gene knockout pigs harboring a human artificial chromosome containing the entire dystrophin gene.

Mammalian artificial chromosomes have enabled the introduction of extremely large amounts of genetic information into animal cells in an autonomously replicating, nonintegrating format. However, the evaluation of human artificial chromosomes (HACs) as novel tools for curing intractable hereditary disorders has been hindered by the limited efficacy of the delivery system. We generated dystrophin gene knockout (DMD-KO) pigs harboring the HAC bearing the entire human DMD via a somatic cell cloning procedure (DYS-HAC-cloned pig). Restored human dystrophin expression was confirmed by immunofluorescence staining in the skeletal muscle of the DYS-HAC-cloned pigs. Viability at the first month postpartum of the DYS-HAC-cloned pigs, including motor function in the hind leg and serum creatinine kinase level, was improved significantly when compared with that in the original DMD-KO pigs. However, decrease in systemic retention of the DYS-HAC vector and limited production of the DMD protein might have caused severe respiratory impairment with general prostration by 3 months postpartum. The results demonstrate that the use of transchromosomic cloned pigs permitted a straightforward estimation of the efficacy of the DYS-HAC carried in affected tissues/organs in a large-animal disease model, providing novel insights into the therapeutic application of exogenous mammalian artificial chromosomes.

A Transgenic Pig Model With Human Mutant SOD1 Exhibits the Early Pathology of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) causes progressive degeneration of the motor neurons. In this study, we delivered the genetic construct including the whole locus of human mutant superoxide dismutase 1 (SOD1) with the promoter region of human SOD1 into porcine zygotes using intracytoplasmic sperm injection-mediated gene transfer, and we thereby generated a pig model of human mutant SOD1-mediated familial ALS. The established ALS pig model exhibited an initial abnormality of motor neurons with accumulated misfolded SOD1. The ALS pig model, with a body size similar to that of human beings, will provide opportunities for cell and gene therapy platforms in preclinical translational research.

Development of a Cryopreservation Technique for Xenogeneic Kidney Grafts: Evaluation Using a Mouse Model.

To align the xeno-metanephros and renal progenitor cell timing for transplantation treatments, cryopreservation techniques and an efficient transportation of regenerated renal products such as xeno-metanephroi and renal progenitor cells should be established. Therefore, we propose a novel method of xenogeneic regenerative medicine for patients with chronic kidney disease by grafting porcine fetal kidneys injected with human renal progenitor cells. To develop a useful cryopreserve system of porcine fetal kidney and human renal progenitor cells, we examined the cryopreservation of a fetal kidney implanted with renal progenitor cells in a mouse model. First, we developed a new method for direct cell injection under the capsule of the metanephros using gelatin as a support for unzipped fetal kidneys. Then, we confirmed in vitro that the nephrons derived from the transplanted cells were regenerated even after cryopreservation before and after cell transplantation. Furthermore, the cryopreserved chimeric metanephroi grew, and regenerated nephrons were observed in NOD. We confirmed that even in cryopreserved chimeric metanephroi, transplanted cell-derived nephrons as well as fresh transplants grew.

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.

Feasibility of large experimental animal models in testing novel therapeutic strategies for diabetes.

Diabetes is among the top 10 causes of death in adults and caused approximately four million deaths worldwide in 2017. The incidence and prevalence of diabetes is predicted to increase. To alleviate this potentially severe situation, safer and more effective therapeutics are urgently required. Mice have long been the mainstay as preclinical models for basic research on diabetes, although they are not ideally suited for translating basic knowledge into clinical applications. To validate and optimize novel therapeutics for safe application in humans, an appropriate large animal model is needed. Large animals, especially pigs, are well suited for biomedical research and share many similarities with humans, including body size, anatomical features, physiology, and pathophysiology. Moreover, pigs already play an important role in translational studies, including clinical trials for xenotransplantation. Progress in genetic engineering over the past few decades has facilitated the development of transgenic animals, including porcine models of diabetes. This article discusses features that attest to the attractiveness of genetically modified porcine models of diabetes for testing novel treatment strategies using recent technical advances.

Genetically engineered pigs manifesting pancreatic agenesis with severe diabetes.

INTRODUCTION: Pancreatic duodenum homeobox 1 (Pdx1) expression is crucial for pancreatic organogenesis and is a key regulator of insulin gene expression. Hairy and enhancer of split 1 (Hes1) controls tissue morphogenesis by maintaining undifferentiated cells. Hes1 encodes a basic helix loop helix (bHLH) transcriptional repressor and functionally antagonizes positive bHLH genes, such as the endocrine determination gene neurogenin-3. Here, we generated a new pig model for diabetes by genetic engineering Pdx1 and Hes1 genes. RESEARCH DESIGN AND METHODS: A transgenic (Tg) chimera pig with germ cells carrying a construct expressing Hes1 under the control of the Pdx1 promoter was used to mate with wild-type gilts to obtain Tg piglets. RESULTS: The Tg pigs showed perinatal death; however, this phenotype could be rescued by insulin treatment. The duodenal and splenic lobes of the Tg pigs were slender and did not fully develop, whereas the connective lobe was absent. ß cells were not detected, even in the adult pancreas, although other endocrine cells were detected, and exocrine cells functioned normally. The pigs showed no irregularities in any organs, except diabetes-associated pathological alterations, such as retinopathy and renal damage. CONCLUSION: Pdx1-Hes1 Tg pigs were an attractive model for the analysis of pancreatic development and testing of novel treatment strategies for diabetes.

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.

DNA methylation ambiguity in the Fibrillin-1 (FBN1) CpG island shore possibly involved in Marfan syndrome.

Fibrillin-1 (FBN1) is responsible for haploinsufficient and autosomal dominant Marfan syndrome. Even in the same Marfan pedigree, penetrance and expressivity in heterozygous individuals can differ and result in variable disease onset and severity. Thus, other factors in addition to mutations in FBN1 are likely to contribute to the disease. In this study, we examined the regulation of FBN1 in porcine Marfan syndrome model, focusing on DNA methylation patterns distinguishable as wild-type (WT) and FBN1 null (KO) alleles in heterozygous cells. Most importantly, the ratio of the transcriptionally active hypomethylated WT allele was altered during cellular passage and highly correlated with FBN1 mRNA level compared with that in the KO allele. Transcribed FBN1 RNA from the KO allele was abolished after splicing coupled with translational initiation, suggesting that the functional FBN1 mRNA levels were affected by DNA methylation of the WT allele.

Correction: Pigs with δ-sarcoglycan deficiency exhibit traits of genetic cardiomyopathy.

This article was originally published under Nature Research's License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the article have been modified accordingly.

Pigs with δ-sarcoglycan deficiency exhibit traits of genetic cardiomyopathy.

Genetic cardiomyopathy is a group of intractable cardiovascular disorders involving heterogeneous genetic contribution. This heterogeneity has hindered the development of life-saving therapies for this serious disease. Genetic mutations in dystrophin and its associated glycoproteins cause cardiomuscular dysfunction. Large animal models incorporating these genetic defects are crucial for developing effective medical treatments, such as tissue regeneration and gene therapy. In the present study, we knocked out the δ-sarcoglycan (δ-SG) gene (SGCD) in domestic pig by using a combination of efficient de novo gene editing and somatic cell nuclear transfer. Loss of δ-SG expression in the SGCD knockout pigs caused a concomitant reduction in the levels of α-, ß-, and γ-SG in the cardiac and skeletal sarcolemma, resulting in systolic dysfunction, myocardial tissue degeneration, and sudden death. These animals exhibited symptoms resembling human genetic cardiomyopathy and are thus promising for use in preclinical studies of next-generation therapies.

Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation.

We have previously established a concept of developing exogenic pancreas in a genetically modified pig fetus with an apancreatic trait, thereby proposing the possibility of in vivo generation of functional human organs in xenogenic large animals. In this study, we aimed to demonstrate a further proof-of-concept of the compensation for disabled organogeneses in pig, including pancreatogenesis, nephrogenesis, hepatogenesis, and vasculogenesis. These dysorganogenetic phenotypes could be efficiently induced via genome editing of the cloned pigs. Induced dysorganogenetic traits could also be compensated by allogenic blastocyst complementation, thereby proving the extended concept of organ regeneration from exogenous pluripotent cells in empty niches during various organogeneses. These results suggest that the feasibility of blastocyst complementation using genome-edited cloned embryos permits experimentation toward the in vivo organ generation in pigs from xenogenic pluripotent cells.

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.

Distributions of endocrine cell clusters during porcine pancreatic development.

BACKGROUND: Pancreatic islet xenotransplantation is a potential treatment for diabetes mellitus, and porcine pancreas may provide a readily available source of islets. Islets in juvenile pigs are smaller than those in young adult pigs, but the insulin content is very similar. In addition, as juvenile pigs are more easily reared in uncontaminated conditions, many researchers have conducted studies using pancreatic islets from juvenile pigs. We aimed to analyze the distributions of endocrine cell clusters by comprehensively evaluating juvenile porcine pancreatic development and to propose an appropriate age at which islets could be isolated from the juvenile porcine pancreas. METHODS: Splenic (SL) and duodenal lobe (DL) samples were collected from the pancreases of pigs aged 0-180 days (n = 3/day after birth). The chronological changes in endocrine cell clustering were analyzed in relation to morphological changes, cell characterization, numbers, islet areas, and gene expression. RESULTS: In juvenile pigs aged 0-21 days, the pancreas contained numerous endocrine cells, and compact islets appeared from 21 days of age. Well-defined small islets were seen at 28 days of age, and the clusters were denser in the SL than in the DL. At 35 days of age, the islets were morphologically similar to those observed at 180 days of age, and the greater number of islets was similar to that seen at 90 days of age. The differences in the islets' cytoarchitecture between the lobes were negligible. The expression of ß-cell-related genes was higher in the juvenile pancreas than in the adult pancreas, and the expression of neurogenin-3 decreased dramatically over time. CONCLUSIONS: These findings may have implications for attempts to refine the most appropriate age for islet isolation from porcine donors. Focusing on porcine pancreatic islets isolated at around 35 days after birth may offer benefits regarding their xenotransplantation potential.

The phenotype of a pig with monosomy X resembling Turner syndrome symptoms: a case report.

The partial or complete loss of one X chromosome in humans causes Turner syndrome (TS), which is accompanied by a range of physical and reproductive pathologies. This article reports similarities between the phenotype of a pig with monosomy X and the symptoms of TS in humans. Born as the offspring of a male pig carrying a mutation in an X-chromosomal gene, ornithine transcarbamylase (OTC), the female pig (37,XO) was raised to the age of 36 months. This X-monosomic pig presented with abnormal physical characteristics including short stature, micrognathia, and skeletal abnormalities in the limbs. Furthermore, the female did not exhibit an estrous cycle, even after reaching the age of sexual maturity, and showed no ovarian endocrine activity except for an irregular increase in blood 17ß-estradiol levels, which was seemingly attributable to sporadic follicular development. An autopsy at 36 months revealed an undeveloped reproductive tract with ovaries that lacked follicles. These data demonstrated that the growth processes and anatomical and physiological characteristics of an X-monosomic pig closely resembled those of a human with TS.