Pig models for translational Duchenne muscular dystrophy research.

Duchenne muscular dystrophy (DMD) is caused by mutations in the X-linked DMD gene, resulting in the absence of dystrophin, progressive muscle degeneration, and heart failure. Genetically tailored pig models resembling human DMD mutations recapitulate the biochemical, clinical, and pathological hallmarks of DMD with an accelerated disease progression compared to human patients. DMD pigs have been used to evaluate therapeutic concepts such as gene editing to reframe a disrupted DMD reading frame or the delivery of artificial chromosome vectors carrying the complete DMD gene. Moreover, DMD pigs have been instrumental in validating new diagnostic modalities such as multispectral optoacoustic tomography (MSOT) for non-invasive monitoring of disease progression. DMD pigs may thus help to bridge the gap between proof-of-concept studies in cellular or rodent models and clinical studies in patients.

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.

Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction.

Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.

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.

The hybrid CL-SP-D molecule has the potential to regulate xenogeneic rejection by human neutrophils more efficiently than CD47.

OBJECTIVE: Innate immunity plays a vital role in xenotransplantation. A CD47 molecule, binding to the SIRPα expressed on monocyte/macrophage cells, can suppress cytotoxicity. Particularly, the SIRPα contains ITIM, which delivers a negative signal. Our previous study demonstrated that the binding between CL-P1 and surfactant protein-D hybrid (CL-SP-D) with SIRPα regulates macrophages' phagocytic activity. In this study, we examined the effects of human CD47 and CL-SP-D expression on the inhibition of xenograft rejection by neutrophils in swine endothelial cells (SECs). METHODS: We first examined SIRPα expression on HL-60 cells, a neutrophil-like cell line, and neutrophils isolated from peripheral blood. CD47-expressing SECs or CL-SP-D-expressing SECs were generated through plasmid transfection. Subsequently, these SECs were co-cultured with HL-60 cells or neutrophils. After co-culture, the degree of cytotoxicity was calculated using the WST-8 assay. The suppressive function of CL-SP-D on neutrophils was subsequently examined, and the results were compared with those of CD47 using naïve SECs as controls. Additionally, we assessed ROS production and neutrophil NETosis. RESULTS: In initial experiments, the expression of SIRPα on HL-60 and neutrophils was confirmed. Exposure to CL-SP-D significantly suppressed the cytotoxicity in HL-60 (p = 0.0038) and neutrophils (p = 0.00003). Furthermore, engagement with CD47 showed a suppressive effect on neutrophils obtained from peripheral blood (p = 0.0236) but not on HL-60 (p = 0.4244). The results of the ROS assays also indicated a significant downregulation of SEC by CD47 (p = 0.0077) or CL-SP-D (p = 0.0018). Additionally, the suppression of NETosis was confirmed (p = 0.0125) in neutrophils co-cultured with S/CL-SP-D. CONCLUSION: These results indicate that CL-SP-D is highly effective on neutrophils in xenogeneic rejection. Furthermore, CL-SP-D was more effective than CD47 at inhibiting neutrophil-mediated xenograft rejection.

Effect of microgravity on mammalian embryo development evaluated at the International Space Station.

Mammalian embryos differentiate into the inner cell mass (ICM) and trophectoderm at the 8-16 cell stage. The ICM forms a single cluster that develops into a single fetus. However, the factors that determine differentiation and single cluster formation are unknown. Here we investigated whether embryos could develop normally without gravity. As the embryos cannot be handled by an untrained astronaut, a new device was developed for this purpose. Using this device, two-cell frozen mouse embryos launched to the International Space Station were thawed and cultured by the astronauts under microgravity for 4 days. The embryos cultured under microgravity conditions developed into blastocysts with normal cell numbers, ICM, trophectoderm, and gene expression profiles similar to those cultured under artificial-1 g control on the International Space Station and ground-1 g control, which clearly demonstrated that gravity had no significant effect on the blastocyst formation and initial differentiation of mammalian embryos.

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.

Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD.

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.

Cryopreservation of Fetal Porcine Kidneys for Xenogeneic Regenerative Medicine.

Kidney xenotransplantation has been attracting attention as a treatment option for end-stage renal disease. Fetal porcine kidneys are particularly promising grafts because they can reduce rejection through vascularization from host vessels. We are proposing xenogeneic regenerative medicine using fetal porcine kidneys injected with human nephron progenitor cells. For clinical application, it is desirable to establish reliable methods for the preservation and quality assessment of grafts. We evaluated the differentiation potency of vitrified porcine fetal kidneys compared with nonfrozen kidneys, using an in vivo differentiation model. Fetal porcine kidneys connected to the bladder were frozen via vitrification and stored in liquid nitrogen. Several days later, they were thawed and transplanted under the retroperitoneum of immunocompromised mice. After 14 days, the frozen kidneys grew and differentiated into mature nephrons, and the findings were comparable to those of nonfrozen kidneys. In conclusion, we demonstrated that the differentiation potency of vitrified fetal porcine kidneys could be evaluated using this model, thereby providing a practical protocol to assess the quality of individual lots.

Genome Editing of Pig.

Pigs have anatomical and physiological characteristics similar to humans; therefore, genetically modified pigs have the potential to become a valuable bioresource in biomedical research. In fact, considering the increasing need for translational research, pigs are useful for studying intractable diseases, organ transplantation, and regenerative medicine as large-scale experimental animals with excellent potential for extrapolation to humans. With the advent of zinc finger nucleases (ZFNs), breakthroughs in genome editing tools such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) have facilitated the efficient generation of genetically modified pigs. Genome editing has been used in pigs for more than 10 years; now, along with knockout pigs, knock-in pigs are also gaining increasing importance. In this chapter, we describe the establishment of gene-modified cells (nuclear donor cells), which are necessary for gene knockout and production of knock-in pigs via somatic cell nuclear transplantation, as well as the production of gene knockout pigs using a simple cytoplasmic injection method.

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.

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station.

Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. The recovery rate of embryos was over 90%, and its developmental rate to the blastocyst were over 80%. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at -80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although the recovery rate of embryos after thawing were not high (24%-78%) and embryonic development in ETC could not be observed, thawed embryos formed blastocysts after 4 days of culture (29%-100%) without direct contact. Thus, this ETC could be used for untrained astronauts to thaw and culture frozen embryos on the ISS. In addition, this ETC will be an important advance in fields such as clinical infertility and animal biotechnology when recovery rate of embryos were improved nearly 100%.

Chondrocranial variation in chicken domestication.

The chondrocranium is a key structure of the skull, but our knowledge of its embryonic development is based mostly on investigations of few stages across taxa. Variation of chondrocranial features is known across species, but little is known about intraspecific variation, or its evolution in the context of domestication. Here, we investigated two specific structures of the chondrocranium in three windows of embryonic development. The anatomy of one of these structures was also compared among adult skulls of chickens and their wild ancestor (red junglefowl [RJF]). The proccesus tectalis and the prenasal process, along with the surrounding area of the orbitonasal foramina, presented variation throughout the ontogeny and in the adults. The processus tectalis showed distinct variation from the earliest stage studied to the adult. The numbers of orbitonasal foramina were also found to be variable in the ancestor and breeds studied. Furthermore, during early embryonic development, the prenasal process is similar across breeds and RJF, but later in embryonic development this structure presents variable states. The embryonic and adult variation found herein could be an example of intraspecific variation under domestication, resulting from different types of tissue interrelationship during development.

Moving towards a novel therapeutic strategy for hyperammonemia that targets glutamine metabolism.

Patients with urea cycle disorders intermittently develop episodes of decompensation with hyperammonemia. Although such an episode is often associated with starvation and catabolism, its molecular basis is not fully understood. First, we attempted to elucidate the mechanism of such starvation-associated hyperammonemia. Using a mouse embryonic fibroblast (MEF) culture system, we found that glucose starvation increases ammonia production, and that this increase is associated with enhanced glutaminolysis. These results led us to focus on α-ketoglutarate (AKG), a glutamate dehydrogenase inhibitor, and a major anaplerotic metabolite. Hence, we sought to determine the effect of dimethyl α-ketoglutarate (DKG), a cell-permeable AKG analog, on MEFs and found that DKG mitigates ammonia production primarily by reducing flux through glutamate dehydrogenase. We also verified that DKG reduces ammonia in an NH4 Cl-challenged hyperammonemia mouse model and observed that DKG administration reduces plasma ammonia concentration to 22.8% of the mean value for control mice that received only NH4 Cl. In addition, we detected increases in ornithine concentration and in the ratio of ornithine to arginine following DKG treatment. We subsequently administered DKG intravenously to a newborn pig with hyperammonemia due to ornithine transcarbamylase deficiency and found that blood ammonia concentration declined significantly over time. We determined that this effect is associated with facilitated reductive amination and glutamine synthesis. Our present data indicate that energy starvation triggers hyperammonemia through enhanced glutaminolysis and that DKG reduces ammonia accumulation via pleiotropic mechanisms both in vitro and in vivo. Thus, cell-permeable forms of AKG are feasible candidates for a novel hyperammonemia treatment.

Pig models for Duchenne muscular dystrophy - from disease mechanisms to validation of new diagnostic and therapeutic concepts.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal muscles and heart. Animal models are essential for preclinical evaluation of novel diagnostic procedures and treatment strategies. Gene targeting/editing offers the possibility of developing tailored pig models for monogenic diseases. The first porcine DMD model was generated by deletion of DMD exon 52 (DMDΔ52) in cultured kidney cells, which were used for somatic cell nuclear transfer to produce DMDΔ52 offspring. The animals resembled clinical, biochemical, and pathological hallmarks of DMD, but died before sexual maturity, thus preventing their propagation by breeding. This limitation was overcome by the generation of female heterozygous DMDΔ52 carrier pigs, which allowed the establishment of a large breeding colony. In this overview, we summarize how porcine DMD models have been used for dissecting disease mechanisms, for validating multispectral optoacoustic tomography as an imaging modality for monitoring fibrosis, and for preclinical testing of a CRISPR/Cas9 based approach to restore an intact DMD reading frame. Particular advantages of porcine DMD models include their targeted design and the rapid disease progression with early cardiac involvement, facilitating translational studies in reasonable time frames.

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.

Aspects of the Complement System in New Era of Xenotransplantation.

After producing triple (Gal, H-D and Sda)-KO pigs, hyperacute rejection appeared to no longer be a problem. However, the origin of xeno-rejection continues to be a controversial topic, including small amounts of antibodies and subsequent activation of the graft endothelium, the complement recognition system and the coagulation systems. The complement is activated via the classical pathway by non-Gal/H-D/Sda antigens and by ischemia-reperfusion injury (IRI), via the alternative pathway, especially on islets, and via the lectin pathway. The complement system therefore is still an important recognition and effector mechanism in xeno-rejection. All complement regulatory proteins (CRPs) regulate complement activation in different manners. Therefore, to effectively protect xenografts against xeno-rejection, it would appear reasonable to employ not only one but several CRPs including anti-complement drugs. The further assessment of antigens continues to be an important issue in the area of clinical xenotransplantation. The above conclusions suggest that the expression of sufficient levels of human CRPs on Triple-KO grafts is necessary. Moreover, multilateral inhibition on local complement activation in the graft, together with the control of signals between macrophages and lymphocytes is required.