A Comparison Between GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;TBM Pig Kidneys Transplanted in Nonhuman Primates.

Because there is a shortage of donor kidneys, researchers are exploring the possibility of using genetically modified pig kidneys for transplantation. Approaches involving knockout of carbohydrate genes or knockin of protective proteins have been attempted to determine the best gene modifications. In this study, we utilized GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;thrombomodulin (TBM) pigs for transplantation in nonhuman primates (NHPs). The NHPs survived for 4 weeks after kidney transplantation (4 WAT) from the GalT-/-;hCD39;hCD55 pig and for 6 WAT from the GalT-/-;hCD39;hCD46;hCD55;TBM pig. However, messenger RNA (mRNA) sequencing and immunohistochemistry analysis revealed that the 6 WAT kidney exhibited more severe apoptosis, inflammation, loss of renal function, and renal fibrosis than the 4 WAT kidney. These results indicate that additional knockin of complement regulator (hCD46) and coagulation regulator (TBM) is not enough to prevent renal damage, suggesting that improved immune suppression is needed for more prolonged survival.

Construction and validation of a prediction model for public acceptance of kidney xenotransplantation in China / 器官移植

Objective To explore the public attitude towards kidney xenotransplantation in China by constructing and validating the prediction model based on xenotransplantation questionnaire. Methods A convenient sampling survey was conducted among the public in China with the platform of Wenjuanxing to analyze public acceptance of kidney xenotransplantation and influencing factors. Using random distribution method, all included questionnaires (n=2 280) were divided into the training and validation sets according to a ratio of 7:3. A prediction model was constructed and validated. Results A total of 2 280 questionnaires were included. The public acceptance rate of xenotransplantation was 71.3%. Multivariate analysis showed that gender, marital status, resident area, medical insurance coverage, religious belief, vegetarianism, awareness of kidney xenotransplantation and whether on the waiting list for kidney transplantation were the independent influencing factors for public acceptance of kidney xenotransplantation (all P<0.05). The area under the curve (AUC) of receiver operating characteristic (ROC) of the prediction model in the training set was 0.773, and 0.785 in the validation set. The calibration curves in the training and validation sets indicated that the prediction models yielded good prediction value. Decision curve analysis (DCA) suggested that the prediction efficiency of the model was high. Conclusions In China, public acceptance of kidney xenotransplantation is relatively high, whereas it remains to be significantly enhanced. The prediction model based on questionnaire survey has favorable prediction efficiency, which provides reference for subsequent research.

Isolation of bone marrow mesenchymal stem cells in transgenic pigs and co-culture with porcine islets / 器官移植

Objective To investigate the isolation and culture of porcine bone marrow mesenchymal stem cell (BMSC) with α-1, 3-galactosyltransferase (GGTA1) gene knockout (GTKO), GTKO/ human CD46 (hCD46) insertion and cytidine monopho-N-acetylneuraminic acid hydroxylase (CMAH)/GGTA1 gene knockout (Neu5GC/Gal), and the protective effect of co-culture with porcine islets on islet cells. Methods Bone marrow was extracted from different transgenic pigs modified with GTKO, GTKO/hCD46 and Neu5GC/Gal. Porcine BMSC were isolated by the whole bone marrow adherent method and then cultured. The morphology of BMSC was observed and the surface markers of BMSC were identified by flow cytometry. Meantime, the multi-directional differentiation induced by BMSC was observed, and the labeling and tracing of BMSC were realized by green fluorescent protein (GFP) transfection. The porcine BMSC transfected with GFP were co-cultured with porcine islet cells. Morphological changes of porcine islet cells were observed, and compared with those in the porcine islet cell alone culture group. Results BMSC derived from pigs were spindle-shaped in vitro, expressing biomarkers of CD29, CD44, CD73, CD90, CD105 and CD166 rather than CD34 and CD45. These cells were able to differentiate into adipocytes, osteoblasts and chondrocytes. Porcine BMSC with GFP transfection could be labeled and traced, which could be stably expressed in the daughter cells after cell division. Porcine BMSC exerted certain protective effect on islet cells. Conclusions GFP-labeled porcine BMSC modified with GTKO, GTKO/hCD46 and Neu5GC/Gal are successfully established, which exert certain protective effect upon islet cells.

Progress in subclinical research of kidney xenotransplantation / 器官移植

Xenotransplantation is an efficient pathway to solve the problem of transplant organ source deficiency in clinical settings. With the increasing progress of gene editing technique and immune suppression regimen, important development has been achieved on researches regarding pig to non-human primate kidney xenotransplantation, which provides a good condition for the introduction of the technique in the clinical application. In view of the substantial difference between human and non-human primate, and to meet the needs of current ethic requirements, it is necessary to perform subclinical studies for pig to human kidney xenotransplantation. In recent years, such subclinical studies with regard to the genetically modified pig to brain death recipient kidney xenotransplantation had been performed, indicating that kidney xenotransplantation gradually began to transit to the clinical development stage. However, donor/recipient selection and immune suppression regimen has not reached a consensus yet, and has to be clarified in subclinical studies. In this article, the current status and confronted problems of donor/recipient selection, immune suppression regimen and post transplantation management in the subclinical studies of kidney xenotransplantation were reviewed, aiming to promote the clinical transformation of kidney xenotransplantation to the clinical application.

Present situation and progress of xenotransplantation at home and abroad / 器官移植

Organ shortage has become one of the major challenges hindering the development of organ transplantation. Xenotransplantation is one of the most valuable methods to resolve global organ shortage. In recent years, the development of genetic engineering technique and research and development of new immunosuppressant have provided novel theoretical basis for xenotransplantation. International scholars have successively carried out researches on xenotransplantation in genetically modified pigs to non-human primates or brain death recipients, making certain substantial progresses. However, most of the researches are still in the preclinical stage, far from clinical application. Therefore, according to the latest preclinical experimental research progress at home and abroad, the history of xenotransplantation, the development of gene modification technology, xenotransplantation rejection and immunosuppression regimens were reviewed, aiming to provide reference for subsequent research of xenotransplantation, promote clinical application of xenotransplantation and bring benefits to more patients with end-stage diseases.

Research report of living donor kidney harvesting in Bama miniature pigs with six gene modified / 器官移植

Objective To summarize the experience and practical value of living donor kidney harvesting in Bama miniature pigs with six gene modified. Methods The left kidney of Bama miniature pigs with six gene modified was obtained by living donor kidney harvesting technique. First, the ureter was occluded, and then the inferior vena cava and abdominal aorta were freed. During the harvesting process, the ureter, renal vein and renal artery were exposed and freed in sequence. The vascular forceps were used at the abdominal aorta and inferior vena cava, and the renal artery and vein were immediately perfused with 4℃ renal preservation solution, and stored in ice normal saline for subsequent transplantation. Simultaneously, the donor abdominal aorta and inferior vena cava gap were sutured. The operation time, blood loss, warm and cold ischemia time, postoperative complications and the survival of donors and recipients were recorded. Results The left kidney of the genetically modified pig was successfully harvested. Intraoperative bleeding was 5 mL, warm ischemia time was 45 s, and cold ischemia time was 2.5 h. Neither donor nor recipient pig received blood transfusion, and urinary function of the kidney transplanted into the recipient was recovered. The donor survived for more than 8 months after the left kidney was resected. Conclusions Living donor kidney harvesting is safe and reliable in genetically modified pigs. Branch blood vessels could be processed during kidney harvesting, which shortens the process of kidney repair and the time of cold ischemia. Living donor kidney harvesting contributes to subsequent survival of donors and other scientific researches.

Application of xenotransplantation in clinical practice / 器官移植

Organ transplantation is the optimal treatment for end-stage organ failure. Nevertheless, organ shortage is a global problem, which limits further development of organ transplantation. Recent research shows that genetically modified pig may become a realistic alternative source of clinical organ transplantation donor. Xenotransplantation may serve as one of the effective measures to resolve the problem of organ shortage. Since 2021, 2 cases of living xenotransplantation and 6 cases of xenotransplantation in brain death recipients have been performed worldwide, and phase Ⅰ clinical trial of xenotransplantation has been launched, and the results have exceeded expectations. Therefore, in this article, recent clinical trial results of xenotransplantation in living and brain death recipients were retrospectively analyzed, and scientific, technical and ethical issues related to clinical research of xenotransplantation were illustrated, hoping to provide reference for clinical research of xenotransplantation in China and promote the development of xenotransplantation in clinical practice.

Advances and applications of genetically modified pig models in biomedical and agricultural field.

Compared with rodents, pigs are closer to humans in terms of anatomy, metabolism and physiology, so they are ideal animal models of human diseases and xenotransplantation donors. In addition, as one of the most important livestock in China, pigs are closely related to our lives in terms of breeding improvement, disease prevention and animal welfare. In this review, we mainly summarize the research progress and future application of genetically modified pig models in the fields of xenotransplantation, molecular breeding and human disease models. We wish to take this opportunity to raise the awareness of researchers in related fields on cutting-edge technologies such as gene editing and understand the significance of genetically modified pig models in life science research.

Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes-In Vitro Studies.

Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and α-galactosidase A (hGLA) genes would allow for removal of this obstacle in porcine transgenic epidermal keratinocytes (PEKs). We sought to determine not only the expression profiles of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) proteins, but also the relative abundance (RA) of Galα1→3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2×hGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rhα1,2-FT and rhα-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Galα1→3Gal epitope that were assessed by lectin fluorescence and lectin blotting were found to be significantly diminished in each variant of genetically modified PEK line as compared to those observed in the control nontransgenic PEKs. Notably, the bi-transgenic PEKs were characterized by significantly lessened (but still detectable) RAs of Galα1→3Gal epitopes as compared to those identified for both types of mono-transgenic PEK lines. Additionally, our current investigation showed that the coexpression of two protective transgenes gave rise to enhanced abrogation of Galα→3Gal epitopes in hFUT2×hGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human α-1,2-FT and α-Gal A proteins followed by identification of the extent of abrogating the abundance of Galα1→3Gal epitopes in the ex vivo expanded PEKs stemming from mono- and bi-transgenic pigs were found to be a sine qua non condition for efficiently ex situ protecting stable lines of skin-derived somatic cells inevitable in further studies. The latter is due to be focused on determining epigenomic reprogrammability of single- or double-transgenic cell nuclei inherited from adult cutaneous keratinocytes in porcine nuclear-transferred oocytes and corresponding cloned embryos. To our knowledge, this concept was shown to represent a completely new approach designed to generate and multiply genetically transformed pigs by somatic cell cloning for the needs of reconstructive medicine and dermoplasty-mediated tissue engineering of human integumentary system.

Impact of donor and prolonged cold ischemia time of neonatal pig pancreas on neonatal pig islet transplant outcome.

BACKGROUND: Genetically modified pigs (GMP) have been developed to alleviate the shortage of donors in human islet transplantation and rejection. In this study, we characterized and compared the islets from GalTKO, GalTKO/hCD46, GalTKO/hCD46/hCD39, and wild-type (WT) neonatal pigs. METHODS: Islets were isolated from GMP and WT pig pancreases that have been packaged with ice pack for at least 24 hours. The difference in gene expression and function of islets were evaluated by microarray analysis and transplantation of islets under the kidney capsule of streptozotocin-induced diabetic immune-deficient mice, respectively. Blood glucose levels of these mice were monitored weekly post-transplantation for >100 days, and islet grafts were collected and evaluated for the presence of endocrine cells. RESULTS: The genes involved in extracellular components, cell adhesion, glucose metabolism, and inflammatory response are differentially expressed between GMP and WT pig islets. Variation in the ability of pig islets in correcting the diabetic state of the mouse recipients appears to be dependent on the pig donor. In addition, prolonged cold ischemia time had a negative effect on the transplant outcome. All normoglycemic mice were able to respond well to glucose challenge despite the initial differences in the ability of islet transplants to reverse their diabetic state. Islet xenografts of normoglycemic mice contained abundant insulin- and glucagon-positive cells. CONCLUSION: The effect of GMP and WT neonatal pig islet transplants on hyperglycemia in mice appears to be dependent on the pig donor, and prolonged cold ischemia time negatively affects the neonatal pig islet transplant outcome.

CRISPR/Cas9-Mediated Integration of Large Transgene into Pig CEP112 Locus.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a precise genome manipulating tool that can produce targeted gene mutations in various cells and organisms. Although CRISPR/Cas9 can efficiently generate gene knockout, the gene knock-in (KI) efficiency mediated by homology-directed repair remains low, especially for large fragment integration. In this study, we established an efficient method for the CRISPR/Cas9-mediated integration of large transgene cassette, which carries salivary gland-expressed multiple digestion enzymes (≈ 20 kbp) in CEP112 locus in pig fetal fibroblasts (PFFs). Our results showed that using an optimal homology donor with a short and a long arm yielded the best CRISPR/Cas9-mediated KI efficiency in CEP112 locus, and the targeting efficiency in CEP112 locus was higher than in ROSA26 locus. The CEP112 KI cell lines were used as nuclear donors for somatic cell nuclear transfer to create genetically modified pigs. We found that KI pig (705) successfully expressed three microbial enzymes (ß-glucanase, xylanase, and phytase) in salivary gland. This finding suggested that the CEP112 locus supports exogenous gene expression by a tissue-specific promoter. In summary, we successfully targeted CEP112 locus in pigs by using our optimal homology arm system and established a modified pig model for foreign digestion enzyme expression in the saliva.

[Research progress of producing genetically modified pigs by CRISPR/Cas9 in the medical field].

As pigs are similar to humans in anatomy, physiology and pathology, nutrition metabolism and disease characteristics, genetically modified pigs are already used for the studies of disease mechanism, pathology and toxicology and the evaluation of drugs. But the production of large modified animals is difficult, cumbersome, time-consuming and costly. With the breakthrough of gene editing technology, clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated 9( Cas9)(CRISPR/Cas9) technology has greatly improved the mutation efficiency, reduced the cost and simplified the steps, and promoted the widespread application of genetically modified pigs. In this paper, the production methods of genetically modified pigs and the research progress of genetically modified pigs by CRISPR/Cas9 in the medical field were reviewed.

Research progress of producing genetically modified pigs by CRISPR/Cas9 in the medical field / 生物医学工程学杂志

As pigs are similar to humans in anatomy, physiology and pathology, nutrition metabolism and disease characteristics, genetically modified pigs are already used for the studies of disease mechanism, pathology and toxicology and the evaluation of drugs. But the production of large modified animals is difficult, cumbersome, time-consuming and costly. With the breakthrough of gene editing technology, clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated 9( Cas9)(CRISPR/Cas9) technology has greatly improved the mutation efficiency, reduced the cost and simplified the steps, and promoted the widespread application of genetically modified pigs. In this paper, the production methods of genetically modified pigs and the research progress of genetically modified pigs by CRISPR/Cas9 in the medical field were reviewed.

A 90-day subchronic study of rats fed lean pork from genetically modified pigs with muscle-specific expression of recombinant follistatin.

Because cardiovascular disease incidence has rapidly increased in recent years, people are choosing relatively healthier diets with low animal fat. A transgenic pig with low fat and a high percentage of lean meat was created in 2011; this pig overexpresses the follistatin (FST) gene. To evaluate the safety of lean pork derived from genetically modified (GM) pigs, a subchronic oral toxicity study was conducted using Sprague-Dawley rats. GM pork and non-GM pork were incorporated into the diet at levels of 3.75%, 7.5%, and 15% (w/w), and the main nutrients of the various diets were subsequently balanced. The safety of GM pork was assessed by comparison of the toxicology response variables in Sprague-Dawley rats consuming diets containing GM pork with those consuming non-GM pork. No treatment-related adverse or toxic effects were observed based on an examination of the daily clinical signs, body weight, food consumption, hematology, serum biochemistry, and organ weight or based on gross and histopathological examination. The results demonstrate that GM pork is as safe for consumption as conventional pork.

Current Strategies for Successful Islet Xenotransplantation / 대한이식학회지

Diabetes mellitus is increasing all over the world and is a serious health problem. Pancreatic islet transplantation is promising treatment for diabetes mellitus, but an imbalance between deceased pancreas donors and recipients limited the widespread clinical application. Therefore, pig islets could be used as an alternative islet source in transplantation. However, a big hurdle to clinical application of islet xenotransplantation is the instant blood mediated inflammatory reaction (IBMIR), which is characterized by activation of the coagulation cascade, platelets and complement systems. Innate immune cells infiltrate the islets in the process of IBMIR and thereby accelerate the early graft loss. Characteristics of IBMIR in islet xenotransplantion are very different from the rejection in solid organ xenotransplantation. Therefore, we focus on the molecules for surmounting IBMIR in order to accomplish successful islet xenotransplantation. To prevent the IBMIR in islet xenotransplantation, development of genetic modified pigs containing anti-coagulant, anti-thrombosis and complement regulatory genes, or capsulation of islet with biomaterials for blocking immune response around islet surface can be tried. Galpha-Gal knockout pigs and the diverse transgenic pigs for complement regulatory protein or anti-coagulant genes have been developed for xenotransplantation. This review summarized on characteristics of rejection in islet xenotransplantation and discusses the strategies for overcoming the rejection.