Xenograft Research Articles

What Genetic Modifications of Source Pigs Are Essential and Sufficient for Cell, Tissue, and Organ Xenotransplantation?

Xenotransplantation of porcine organs has made remarkable progress towards clinical application. A key factor has been the generation of genetically multi-modified source pigs for xenotransplants, protected against immune rejection and coagulation dysregulation. While efficient gene editing tools and multi-cistronic expression cassettes facilitate sophisticated and complex genetic modifications with multiple gene knockouts and protective transgenes, an increasing number of independently segregating genetic units complicates the breeding of the source pigs. Therefore, an optimal combination of essential genetic modifications may be preferable to extensive editing of the source pigs. Here, we discuss the prioritization of genetic modifications to achieve long-term survival and function of xenotransplants and summarise the genotypes that have been most successful for xenogeneic heart, kidney, and islet transplantation. Specific emphasis is given to the choice of the breed/genetic background of the source pigs. Moreover, multimodal deep phenotyping of porcine organs after xenotransplantation into human decedents will be discussed as a strategy for selecting essential genetic modifications of the source pigs. In addition to germ-line gene editing, some of these modifications may also be induced during organ preservation/perfusion, as demonstrated recently by the successful knockdown of swine leukocyte antigens in porcine lungs during ex vivo perfusion.

Grafting semi-wild tomato GZ-05 rootstocks improved cold tolerance via the signalling of melatonin and jasmonic acid

Tomato (Solanum lycopersicum) cultivation in the off-season is significantly hindered by cold stress; hence, utilising stress-resistant rootstocks in grafting is a critical solution. This study used 30 semi-wild tomato GZ-05 plants as rootstocks and Ailsa Craig (AC) tomatoes as scions. After cold stress, the scion tolerance index, leaf ion permeability, and other physiological and biological indicators were used to determine the most tolerant plants. To understand the molecular basis of GZ-05 rootstock cold stress resistance, RNA sequencing and reverse transcription polymerase chain reaction techniques were used to compare the varying genes expressed in the grafted AC/GZ-05 and self-grafted AC/AC plant scion leaves. The results indicated that genes associated with melatonin (MT) and jasmonic acid (JA) production and their signalling pathways were considerably altered. The initial MT and JA levels in the GZ-05-grafted plant scions were high, and when they were exposed to cold stress, the amount of active MT and JA in AC/GZ-05 heterologous grafts were great. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9, we knocked out the MT synthesis gene (SlCOMT14) and JA synthesis gene (SlLoxD) and found that GZ-05 plant cold stress tolerance decreased. External tests were conducted to evaluate the GZ-05 SlCOMT14 and SlLoxD knockout lines. Source spraying with MT and methyl jasmonate showed that knockout strain cold stress tolerance could be recovered. SlICE1, a tomato cold stress tolerance transcription factor, was silenced, resulting in decreased tolerance to MT- and JA-induced cold stress. The MT/JA-inducer of C-repeat binding factor (CBF) expression 1-CBF pathway may be the mechanism by which the semi-wild tomato GZ-05 rootstock confers cold tolerance to plants. This study has uncovered the molecular mechanism by which grafting semi-wild tomato GZ-05 rootstocks increases plant cold tolerance, thereby laying the groundwork for the utilisation of Guizhou's native semi-wild tomato germplasm resources.

Is Allosensitization Detrimental to Pig Organ Xenotransplantation, and Is Xenosensitization Detrimental to Subsequent Organ Allotransplantation? A Debate Organized by the International Xenotransplantation Association (IXA).

This report summarizes the content of a debate sponsored by eGenesis Bio, organized by the International Xenotransplantation Association (IXA), and attended by more than 150 delegates in the context of the IPITA-IXA-CTRMS Joint Congress held in San Diego in October 2023. The debate centered around two important immunological topics relating to xenotransplantation. The first was a debate relating to the statement that "HLA-sensitized patients are at higher risk for rejecting a pig xenograft." Stuart Knechtle provided evidence to support this statement and Massimo Mangiola opposed it. Before the debate, a majority (>80%) of the audience agreed with this statement. After listening to the debate, this percentage was reduced to approximately 60%. The second debated statement was "Recipients of pig xenografts who develop anti-pig antibodies are at higher risk for rejecting a subsequent allograft." This was proposed by A. Joseph Tector and opposed by Léo H. Bühler. Before the debate, once again a majority of the audience (approximately 60%) believed that prior sensitization to a pig xenograft would be detrimental to the survival of a subsequent allograft. However, after listening to the debate, only about 40% believed this statement to be correct. The topics discussed remain complex and answers are not yet conclusive. However, the present evidence suggests that allosensitization may prove detrimental to subsequent xenotransplantation, whilst sensitization to pig antigens may not be detrimental to subsequent allotransplantation.

Treatment of periodontal osseous defect with bio-oss xenograft and biodegradable IGF fibers: A case report

Osseous defect caused due to periodontitis needed to be repaired or regenerated to avoid tooth loss. Bio-oss is made up of 10% porcine collagen and can induce osteoblastic differentiation due to its volumetric stability and out standing osteoconductive properties. Application of growth factors in regenerative procedures have helped to maintain the scaffold and induce several cellular processes. The biodegradable fibers incorporated with IGF growth factor has strong effect on PDL fibroblasts, mitogenesis, and protein synthesis which promotes osteogenesis and cementogenesis. A patient having grade II furcation and intrabony defect was treated and evaluated after 12 months with biodegradable fibers infused with insulin like growth factor and bio oss xeno graft irt 36. Treated site responded well to the use of the bovine bone mineral with good tissue response and an improvement and there was an overall mean probing pocket reduction in the deepest site of osseous defects. The clinical parameters assessed are plaque index, bleeding on brobing, pocket probing depth, clinical attachment level, glickman’s furcation index and RVG at baseline, 6 and 12 months post operatively. Both the bone grafts resulted in better regeneration when compared to base line and 12 months.Bio oss (bovine bone mineral) and biodegradable fibres infused with insulin like growth factor materials are of considerable promise for regeneration with better clinical and radiographic outcomes.

Elimination of GGTA1, CMAH, β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions.

Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic, while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs. Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection (HAR) that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure, in which process the MHC II molecule plays critical roles. Thus, we generate a 4-gene (GGTA1, CMAH, β4GalNT2, and CIITA) knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously. We successfully obtained 4KO piglets with deficiency in all alleles of genes, and at cellular and tissue levels. Additionally, the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping. Piglets have survived for more than one year in the barrier, and also survived for more than 3 months in the conventional environment, suggesting that the piglets without MHC II can be raised in the barrier and then gradually mated in the conventional environment. 4KO piglets have lower immunogenicity, are safe in genomic level, and are easier to breed than the model with both MHC I and II deletion.

Osteoinductive Properties of Autologous Dentin: An Ex Vivo Study on Extracted Teeth.

Over the last decades, a variety of biomaterials, ranging from synthetic products to autologous and heterologous grafts, have been recommended to conserve and regenerate bone tissue after tooth extraction. We conducted a biochemical study on ground extracted teeth that aimed to evaluate the osteoinductive and osteoconductive potential of dentin by assessing the releases of bone morphogenetic protein (BMP-2), osteocalcin (OC) and osteonectin (ON) over time (24 h, 10 days and 28 days). Twenty-six patients, who required the extraction of nonrestorable teeth, were enrolled in the study according to the inclusion criteria, as follows: thirteen young patients 18 to 49 years of age (UNDER 50), and thirteen patients of 50 to 70 years (OVER 50); a total of twenty-six teeth were extracted, ground and analyzed by enzyme-linked immunosorbent assays (ELISA). All ground teeth released BMP-2, OC and ON at each time point; no differences were observed between the UNDER-50 and OVER-50 patients. The results of the study support the use of autologous dentin as osteoinductive material for bone regeneration procedures, irrespective of patients' ages.

Exploring attitudes toward xenotransplantation: A scoping review of healthcare workers, healthcare students, and kidney patients.

Recent advances mean that formal clinical trials of solid organ xenotransplantation are increasingly likely to begin and patients requiring a kidney transplant could be the first participants. Healthcare workers and healthcare students constitute the current and future workforce that will influence public opinion of xenotransplantation. The attitudes of these populations are important to consider before recruitment for formal clinical trials begins. This scoping review was reported according to the PRISMA extensions for scoping reviews checklist and the Joanna Briggs Institute methodology for scoping reviews. The Scopus, PubMed, and ScienceDirect databases were searched to identify articles that studied the attitudes of healthcare workers, healthcare students, or kidney patients toward xenotransplantation. The search generated 816 articles, of which 27 met the eligibility criteria. The studies were conducted in 14 different countries on five different continents. Participants from the 27 studies totaled 29,836-this was constituted of 6,223 (21%) healthcare workers, 21,067 (71%) healthcare students, and 2,546 (8%) kidney patients. All three groups had an overall positive attitude toward xenotransplantation. However, in studies where participants were asked to consider xenotransplantation when the risks and results were not equal to allotransplantation-the overall attitude switched from positive to negative. The results also found that Spanish-speaking populations expressed more favorable views toward xenotransplantation compared to English-speaking populations. The results of this review suggest that while attitudes of the three groups toward xenotransplantation are-on the face of it-positive, this positivity deteriorates when the risks and outcomes are framed in more clinically realistic terms. Only formal clinical trials can determine how the risks and outcomes of xenotransplantation compare to allotransplantation.

Pollen transcriptomic analysis provided insights into understanding the molecular mechanisms underlying grafting-induced improvement in potato fertility.

Heterologous grafting has been proven to be a valid approach to improving potato fertility, especially when grafting potatoes with other Solanaceae family plants. However, the mechanisms underlying grafting-induced improvement in potato fertility are still unknown. In this study, a poor-fertility potato cultivar "Qingshu No. 9" (Q9) was grafted with a tomato cultivar "Zhongyan988" (ZY988) to study the effects of heterologous grafting in the former. The tuber yield was controlled by different grafting and cultivation approaches, and the correlation between tuber yield and pollen vigor was studied. Comparative transcriptomic analysis of the potential mechanisms of pollen in potato scion fertility changes. Grafting with the tomato rootstock effectively promoted the flower and fruit formation in the scion potato and improved its pollen viability by 15%-20%. In addition, a significant negative correlation was observed between the potato tuber yield and pollen viability, suggesting a potential impact on the metabolic regulatory network related to tuber formation. From the comparative transcriptomic analysis between the pollens from Q9 self-grafted plants and Q9-tomato grafting scion, 513 differentially expressed genes (DEGs) were identified. These DEGs were found to be related to gametophyte and pollen development, carbohydrate metabolism, and protein processing. Thus, these DEGs might be involved in improved fertility after reduced tuberization in plants subjected to heterologous grafting. Potato/tomato heterologous grafting significantly improved the pollen viability of scion potatoes and was associated with the absence of potato tubers. Heterologous grafting promotes the transcription of genes related to protein processing, carbohydrate metabolism, and pollen development in pollen cells, resulting in the production of fertile pollen. Our results provided initial clues to understanding the improvement of potato fertility using the heterologous grafting method, which might be a useful tool in assisted potato breeding.

Genetic diversity, growth and heart function of Auckland Island pigs, a potential source for organ xenotransplantation.

One of the prerequisites for successful organ xenotransplantation is a reasonable size match between the porcine organ and the recipient's organ to be replaced. Therefore, the selection of a suitable genetic background of source pigs is important. In this study, we investigated body and organ growth, cardiac function, and genetic diversity of a colony of Auckland Island pigs established at the Center for Innovative Medical Models (CiMM), LMU Munich. Male and female Auckland Island pig kidney cells (selected to be free of porcine endogenous retrovirus C) were imported from New Zealand, and founder animals were established by somatic cell nuclear transfer (SCNT). Morphologically, Auckland Island pigs have smaller body stature compared to many domestic pig breeds, rendering their organ dimensions well-suited for human transplantation. Furthermore, echocardiography assessments of Auckland Island pig hearts indicated normal structure and functioning across various age groups throughout the study. Single nucleotide polymorphism (SNP) analysis revealed higher runs of homozygosity (ROH) in Auckland Island pigs compared to other domestic pig breeds and demonstrated that the entire locus coding the swine leukocyte antigens (SLAs) was homozygous. Based on these findings, Auckland Island pigs represent a promising genetic background for organ xenotransplantation.

Large-Scale Formation and Long-Term Culture of Hepatocyte Organoids From Streamlined In Vivo Genome-Edited GGTA1-/- Pigs for Bioartificial Liver Applications.

Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 1010 primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1±28.1µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems.

Ядерно-цитоплазматическое созревание ооцитов Sus Scrofa Domesticus в условиях сверхнизких температур

Abstract. Vitrification of female gametes is the most important tool for solving the strategic tasks of cellular reproductive technologies in animal husbandry, veterinary medicine and biomedicine: replication of elite individuals (cloning, transgenesis), biodiversity conservation, organ xenotransplantation. The aim of this study was to evaluate the parameters of nuclear cytoplasmic maturation (chromatin status and mitochondrial activity) and developmental competence of donor porcine oocytes subjected to intra- (IOV) or extra-ovarian (EOV) vitrification and cultured in various maturation systems. Methods. Donor pig oocytes (6–8 months old) were vitrified extra- or intraovarially using cryoprotective agents (DMSO, ethylene glycol, trehalose, sucrose). The composition of cryoprotective agents was supplemented with 0.001 % highly dispersed silica nanoparticles (HDSns). Oocytes were thawed in trehalose solutions in TS-199 with 10 % of fetal bovine serum (FBS). Oocytes were cultured for 44 hours in media supplemented with granulosa cells (106 cells per ml of medium) and/or 0.001% of HDSns at 38.5 °C, in an atmosphere of 5 % CO2. Fertilization in vitro was carried out in accordance with the recommendations presented by us earlier [17. p. 17]. The functional activity of mitochondria and the status of chromatin in oocytes were assessed by double staining using MitoTracker Orange CMTMRos and Hoechst 33258 dyes. Results. The level of matured oocytes was 61 % at EOV and 29 % at IOV, and the level of cleavage embryos in EOV was in 11 % (31 % vs. 20 %, P < 0.05) higher than at IOV when HDSns with granulosa cells were added in culture media. The maximum indicators of the functional activity of mitochondria among devitrified oocytes were in the group of EOV oocytes have cultured in media supplemented with HDSns and granulosa cells (152 ± 14.9 μA). Scientific novelty. The protocols for intra- and extra-ovarian vitrification of porcine oocytes have been modernized. Addition to cryoprotective agents and culture media of HDSns and co-culture of devitrified oocytes with granulosa cells significantly increased the fertility rates of devitrified gametes.

The Young Investigator Committee of the International Xenotransplantation Association-Perspective of advancements in the field in 2023.

The 2023 IXA conference, hosted in San Diego, CA, brimmed with excitement against the backdrop of recent innovations in both the pre-clinical and clinical realms with several first-in-human applications of xenotransplantation. The theme, "Pigs are flying," alluded to the adage that xenotransplantation would only become a clinical reality "when pigs fly," suggesting a day that might never come. The event witnessed significant attendance, with 600 participants-the highest in the history of an IXA-IPITA joint congress. Among the attendees were members of the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and corporate sponsors deeply engaged in the field. We summarize the latest topics from the congress, ranging from the pros/cons of decedent models of xenotransplantation and genetic engineering of porcine heart valves, solid organs, and cells for clinical translation and their regulatory and ethical landscape.

The Faculty of Theology Students' Attitudes, Knowledge Levels and Behaviours on Organ Donation and Xenotransplantation

The Faculty of Theology Students' Attitudes, Knowledge Levels and Behaviours on Organ Donation and Xenotransplantation

Pressing ethical issues relating to clinical pig organ transplantation studies.

Clinical pig heart transplant experiments have been undertaken, and further clinical experiments and/or clinical trials of gene-edited pig organ xenotransplantation are anticipated. The ethical issues relating to xenotransplantation have been discussed for decades but with little resolution. Consideration of certain ethical issues is more urgent than others, and the need to attain consensus is important. These issues include: (i) patient selection criteria for expanded access and/or clinical trials; (ii) appropriate protection of the patient from xenozoonoses, that is, infections caused by pig microorganisms transferred with the organ graft, (iii) minimization of the risk of a xenozoonosis to bystanders, and (iv) the need for additional public perception studies. We discuss why it is important and urgent to achieve consensus on these ethical issues prior to carrying out further expanded access experiments or initiating formal clinical trials. The ways forward on each issue are proposed.

Chapter 3 - Autologous vs heterologous cell replacement strategies for Parkinson disease and other neurologic diseases

Successful cell replacement strategies for brain repair depend on graft integration into the neural network, which is affected by the immune response to the grafted cells. Using Parkinson disease as an example, in this chapter, we consider the immune system interaction and its role in autologous vs heterologous graft survival and integration, as well as past and emerging strategies to overcome the immunologic response. We also reflect on the role of nonhuman primate research to assess novel approaches and consider the role of different stakeholders on advancing the most promising new approaches into the clinic.

NECESSITY IN XENOTRANSPLANTATION: ISLAMIC PERSPECTIVES REVISITED

Organ failures cause significant problems and sufferings which affect the livelihood, quality of life and decrease in life expectancy to the person. The caregivers, family and community also carry the disease burden and costs of the organ failures. Allotransplantation (AT) offers probable cures to organ failure but is limited by the supply of human organs. Organ shortages have resulted in numerous biomedical ethical problems and illegal activities. Xenotransplantation (XT) and specifically Porcine Xenotransplantation (PXT) has high expectations from researchers and clinicians to be the alternative to AT. From the Islamic perspectives, multiple deliberations and fatwas are issued that vary from total prohibition to conditional permissibility and full permissibility. Often, ḍarūrah or dire necessity is invoked to make XT or PXT permissible. The relevant biomedical issues and whether pre-conditions for ḍarūrah have been satisfied are discussed.

Current status and challenges of pig-to-human organ xenotransplantation.

Current status and challenges of pig-to-human organ xenotransplantation.

Regulatory macrophages in solid organ xenotransplantation.

Due to a critical organ shortage, pig organs are being explored for use in transplantation. Differences between species, particularly in cell surface glycans, can trigger elevated immune responses in xenotransplantation. To mitigate the risk of hyperacute rejection, genetically modified pigs have been developed that lack certain glycans and express human complement inhibitors. Nevertheless, organs from these pigs may still provoke stronger inflammatory and innate immune reactions than allotransplants. Dysregulation of coagulation and persistent inflammation remain obstacles in the transplantation of pig organs into primates. Regulatory macrophages (Mregs), known for their anti-inflammatory properties, could offer a potential solution. Mregs secrete interleukin 10 and transforming growth factor beta, thereby suppressing immune responses and promoting the development of regulatory T cells. These Mregs are typically induced via the stimulation of monocytes or macrophages with macrophage colony-stimulating factor and interferon gamma, and they conspicuously express the stable marker dehydrogenase/reductase 9. Consequently, understanding the precise mechanisms governing Mreg generation, stability, and immunomodulation could pave the way for the therapeutic use of Mregs generated in vitro. This approach has the potential to reduce the required dosages and durations of anti-inflammatory and immunosuppressive medications in preclinical and clinical settings.

Regulatory macrophages as potential cell-based immunotherapy for organ xenotransplantation

Regulatory macrophages as potential cell-based immunotherapy for organ xenotransplantation

Advances in Organ and Tissue Xenotransplantation.

End-stage organ failure can result from various preexisting conditions and occurs in patients of all ages, and organ transplantation remains its only treatment. In recent years, extensive research has been done to explore the possibility of transplanting animal organs into humans, a process referred to as xenotransplantation. Due to their matching organ sizes and other anatomical and physiological similarities with humans, pigs are the preferred organ donor species. Organ rejection due to host immune response and possible interspecies infectious pathogen transmission have been the biggest hurdles to xenotransplantation's success. Use of genetically engineered pigs as tissue and organ donors for xenotransplantation has helped to address these hurdles. Although several preclinical trials have been conducted in nonhuman primates, some barriers still exist and demand further efforts. This review focuses on the recent advances and remaining challenges in organ and tissue xenotransplantation.