Successful Xenotransplantation Research Articles

Genetically Modified Porcine Kidneys Have Sufficient Tissue Integrity for Use in Pig-to-Human Xenotransplantation.

We sought to determine if genetically modified porcine kidneys used for xenotransplantation had sufficient tissue integrity to support long-term function in a human recipient. Kidney transplantation remains the best available treatment for patients with end-stage kidney disease. However, a shortage of available donor human kidneys prevents many patients from achieving the benefits of transplantation. Xenotransplantation is a potential solution to this shortage. Recent pre-clinical human studies have demonstrated kidneys from genetically modified pig donors can be transplanted without hyperacute rejection and are capable of providing creatinine and other solute clearance. It is unknown whether the porcine kidneys would tolerate the relatively higher resting blood pressure in an adult human recipient compared with the pig donor or non-human primate (NHP) recipients used in translational studies. Furthermore, previous experience in NHPs raised concerns about the tissue integrity of the porcine ureter and post-xenotransplant growth of the porcine kidney. Kidneys recovered from porcine donors with 10 gene edits were transplanted into decedent brain-dead recipients who were not eligible for organ donation. Decedents underwent bilateral native nephrectomy before transplant and were followed for 3 to 7 days. Standard induction and maintenance immunosuppression was used as previously reported. Vital signs, including blood pressure, were recorded frequently. Kidney xenografts were assessed daily, serially biopsied, and were measured at implantation and study completion. Three decedents underwent successful xenotransplantation. Subcapsular hematomas developed, requiring incision of the xenograft capsules to prevent Page kidney. Blood pressures were maintained in a physiologic range for adult humans (median arterial pressures (MAP) 108.5mmHg (Interquartile Range (IQR): 97-114mmHg), 74mmHg (IQR: 71-78mmHg), and 95mmHg (IQR: 88-99mmHg, respectively) and no bleeding complications or aneurysm formation was observed. Serial biopsies were taken from the xenografts without apparent loss of tissue integrity despite the lack of a capsule. Ureteroneocystotomies remained intact without evidence of urine leak. Xenograft growth was observed, but plateaued, in 1 decedent with increased volume of the left and right xenografts by 25% and 26%, respectively, and in the context of human growth hormone levels consistently less <0.1ng/ml and insulin-like growth factor 1 levels ranging from 34-50ng/ml. The findings of this study suggest kidneys from 10-gene edited porcine donors have sufficient tissue integrity to tolerate xenotransplantation into a living human recipient. There was no evidence of anastomotic complications, and the xenografts tolerated needle biopsy without issue. Xenograft growth occurred but plateaued by the study end; further observation and investigation will be required to confirm this finding and elucidate underlying mechanisms.

Future directions for xenotransplantation in lungs.

Advancements in preclinical xenotransplant studies have opened doors for clinical heart and kidney xenotransplantation. This review assesses recent progress in lung xenotransplantation research and its potential clinical implications. The efficacy of the humanized von Willebrand factor in reducing platelet sequestration in ex-vivo and in-vivo lung xenotransplant models was showcased. Combining human tissue factor pathway inhibitor and CD47 expression with selectin and integrin inhibition delayed neutrophil and platelet sequestration. Enhanced expression of human complement regulatory proteins and thrombomodulin in genetically engineered pig lungs improved graft survival by reducing platelet activation and modulating coagulation disruptions. Knocking out the CMAH gene decreased antibody-mediated inflammation and coagulation activation, enhancing compatibility for human transplantation. Furthermore, CMAH gene knockout in pigs attenuated sialoadhesin-dependent binding of human erythrocytes to porcine macrophages, mitigating erythrocyte sequestration and anemia. Meanwhile, in-vivo experiments demonstrated extended survival of xenografts for up to 31 days with multiple genetic modifications and comprehensive treatment strategies. Experiments have uncovered vital insights for successful xenotransplantation, driving further research into immunosuppressive therapy and genetically modified pigs. This will ultimately pave the way for clinical trials designed to improve outcomes for patients with end-stage lung disease.

An Approach to Controlling Inflammation and Coagulation in Pig-to-Baboon Cardiac Xenotransplantation.

Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs. Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2). Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach. Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.

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.

Successful xenotransplantation of testicular cells following fractionated chemotherapy of recipient birds

An essential step in the success of germ cell transplantation is the preparation of the recipient’s testicular environment to increase the availability of stem cell niches. However, most methods for this purpose in birds face serious limitations such as partial germ cell depletion, high toxicity and mortality, or the need to use expensive technologies. Here, we validated a simple and practical technique of transferring quail testicular cells into chicken testes depleted of endogenous spermatozoa by fractioned chemotherapy (20 mg/kg/week busulfan for 5 weeks). This protocol resulted in a very low mortality of the treated day-old chicks and, despite maintenance of androgenic activity, sperm production was decreased by 84.3% at 25 weeks of age. NANOG immunostaining revealed that very few to no germ cells were present following treatment with 20 and 40 mg/kg, respectively. RT-qPCR data also showed that c-MYC and NANOG expression declined in these treatments, but GRFα1 and BID expressions remained unaltered among groups. After xenotransplantation, quail germ cells were immunodetected in chicken testes using a species-specific antibody (QCPN), and quail ovalbumin DNA was found in seminal samples collected from chicken recipients. Together, these data confirm that fractionated administration of busulfan in hatchlings is a practical, effective, and safe protocol to prepare recipient male birds capable of supporting xenogeneic spermatogenesis.

Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.

Endothelial Effects of Simultaneous Expression of Human HO-1, E5NT, and ENTPD1 in a Mouse.

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.

Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report

A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome. Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells. After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes. Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future. The University of Maryland School of Medicine, and the University of Maryland Medical Center.

Renin-angiotensin-aldosterone system function in the pig-to-baboon kidney xenotransplantation model

After pig-to-baboon kidney transplantation, episodes of hypovolemia and hypotension from an unexplained mechanism have been reported. This study evaluated the renin-angiotensin-aldosterone system post–kidney xenotransplantation. Kidneys from genetically-engineered pigs were transplanted into 5 immunosuppressed baboons after the excision of the native kidneys. Immunosuppressive therapy was based on the blockade of the CD40/CD154 costimulation pathway. Plasma renin, angiotensinogen (AGT), angiotensin II (Ang II), aldosterone levels, and urine osmolality and electrolytes were measured in healthy pigs, healthy nonimmunosuppressed baboons, and immunosuppressed baboons with life-supporting pig kidney grafts. After pig kidney transplantation, plasma renin and Ang II levels were not significantly different, although Ang II trended lower, even though plasma AGT and potassium were increased. Plasma aldosterone levels were unchanged. Urine osmolality and sodium concentration were decreased. Even in the presence of increasing AGT and potassium levels, lower plasma Ang II concentrations may be because of reduced, albeit not absent, the reactivity of pig renin to cleave baboon AGT, suggesting an impaired response of the renin-angiotensin-aldosterone system to hypovolemic and hypotensive episodes. The maintenance of aldosterone may be protective. The reduced urine osmolality and sodium concentration reflect the decreased ability of the pig kidney to concentrate urine. These considerations should not prohibit successful clinical pig kidney xenotransplantation.

Laboratory Considerations for Successful Xenotransplantation in Humans.

Laboratory Considerations for Successful Xenotransplantation in Humans.

SARS-CoV-2 does not infect pigs, but this has to be verified regularly.

For successful xenotransplantation, freedom of the xenocraft donor from certain viral infections that may harm the organ recipient is important. A novel human coronavirus (CoV) with a respiratory tropism, designated as SARS‐CoV‐2, was first identified in January 2020 in China, but likely has been circulating unnoticed for some time before. Since then, this virus has reached most inhabited areas, resulting in a major global pandemic which is still ongoing. Due to a high number of subclinical infections, re‐infections, geographic differences in diagnostic tests used, and differences in result reporting programs, the percentage of the population infected with SARS‐CoV‐2 at least once has been challenging to estimate. With continuous ongoing infections in people and an overall high viral load, it makes sense to look into possible viral spillover events in pets and farm animals, who are often in close contact with humans. The pig is currently the main species considered for xenotransplantation and hence there is interest to know if pigs can become infected with SARS‐CoV‐2 and if so what the infection dynamics may look like. This review article summarizes the latest research findings on this topic. It would appear that pigs can currently be considered a low risk species, and hence do not pose an immediate risk to the human population or xenotransplantation recipients per se. Monitoring the ever‐changing SARS‐CoV‐2 variants appears important to recognize immediately should this change in the future.

Current Aspects of Xenotransplantation in Human

Xenotransplantation is an approach which will be able to support the increasing demand of organ donation since the organ does not need to come from humans. Up till now, xenotransplantation is still barely performed since its risk and chance of success are still not proven to be safe. One of the biggest obstacles is rejection which may lead to xenograft failure and can occur in certain types and from certain causes. However, the risk of rejection can be minimized by the process of genetic engineering and applying anti-rejection drugs in which scientists and researchers are developing so that the procedure becomes safer in the future. Although the patient who was considered as the first successful xenotransplantation in human case had died a few months after the surgery and the cause of death still remains unsolved, the field of xenotransplantation still keeps developing by researchers, medical universities, and biotechnology companies since they all agree that xenotransplantation will produce lots of advantages and will be a big step of the medical treatment field. Despite the fact that xenotransplantation is a procedure that is risky and raises public concerns and ethical issues, the procedure is still believed to give more benefits towards the patient and medical development since serious problems like organ shortage and high number of deaths on the waiting list will be solved.

Ethical considerations during a pioneering surgical procedure: porcine cardiac xenotransplantation.

Preclinical advances in life-sustaining porcine cardiac xenotransplantation from donor pigs to baboons have paved the way for the performance of porcine cardiac xenotransplantation in a human. This procedure was performed with emergency use authorisation granted by the United States Food and Drug Administration under the umbrella of investigational new drug use on compassionate grounds. The patient was denied candidacy for durable mechanical circulatory support and heart transplantation as a result of non-adherence to medical advice. Successful porcine cardiac xenotransplantation in humans will significantly increase the availability of potential donor organs for long-term management of end-stage heart failure. Human porcine cardiac xenotransplantation is associated with ethical conflicts encompassing multiple ethical principles which are not mutually exclusive and are sometimes conflicting. This article focuses on some of the ethical conflicts encountered in relation to the use of mechanical circulatory support, pretransplant evaluation, shared decision making during informed consent, infectious disease risk, preclinical and clinical testing, and the role of regulatory bodies during performance of the first human porcine cardiac xenotransplantation. An increase in human trials of xenotransplantation procedures is imminent. Potential ethical conflicts associated with xenotransplantation should be addressed appropriately.

Progress in Xenotransplantation: Immunologic Barriers, Advances in Gene Editing, and Successful Tolerance Induction Strategies in Pig-To-Primate Transplantation.

Organ transplantation is the most effective treatment for end stage organ failure, but there are not enough organs to meet burgeoning demand. One potential solution to this organ shortage is xenotransplantation using pig tissues. Decades of progress in xenotransplantation, accelerated by the development of rapid genome editing tools, particularly the advent of CRISPR-Cas9 gene editing technologies, have enabled remarkable advances in kidney and heart xenotransplantation in pig-to-nonhuman primates. These breakthroughs in large animal preclinical models laid the foundation for three recent pig-to-human transplants by three different groups: two kidney xenografts in brain dead recipients deemed ineligible for transplant, and one heart xenograft in the first clinical grade study of pig-to-human transplantation. However, despite tremendous progress, recent data including the first clinical case suggest that gene-modification alone will not overcome all xenogeneic immunologic barriers, and thus an active and innovative immunologic strategy is required for successful xenotransplantation. This review highlights xenogeneic immunologic barriers, advances in gene editing, and tolerance-inducing strategies in pig-to-human xenotransplantation.

Clinical xenotransplantation seems close: Ethical issues persist.

Scientific barriers that have prevented successful xenotransplantation are being breached, yet many ethical issues remain. Some are broad issues that accompany the adoption of novel and expensive technologies, and some are unique to xenotransplantation. Major ethical questions include areas such as: viral transmission; zoonoses and lifetime surveillance; interfering with nature; efficacy, access, and expense; treatment of animals; regulation and oversight.

Long-term dexamethasone treatment increases the engraftment efficiency of human breast cancer cells in adult zebrafish

The host immune system tends to reject xenogenic-implanted cells making tumor development in adult host animal models difficult. Immune system suppression is used for successful xenotransplantation of human cancer cells in many animal models. The studies of cancer development processes in vivo offer opportunities to understand cancer biology and discover new therapeutic strategies. In this context, zebrafish is a model that has been widely applied in the study of human diseases, such as cancer. However, the long-term immunosuppression of these adult zebrafish is still under study as a xenograft animal model for human cancer. This work aimed to evaluate the effects of 21 days of (long-term) exposure of dexamethasone in zebrafish-transplanted with MGSO-3 cells, human breast tumor cell line. Our results show that the animals, while kept on dexamethasone treatment, remained with a 50% reduction in the number of peripheral lymphocytes. In vitro data demonstrated that up to 7 days of dexamethasone treatment did not alter the morphology, proliferation, or viability of MGSO-3 cells. The animals that received a prolonged dexamethasone treatment allowed the engraftment of tumor cells in 100% of the zebrafish tested. These animals also showed tumor progression over 21 days. The experimental group that received only previous exposure to dexamethasone had their tumors regressed after 14 days. In conclusion, the prolonged use of dexamethasone in zebrafish showed a potential strategy for in vivo monitoring of xenograft tumor growth for development studies, as well as in anticancer drug discovery.

Cell-Penetrating Peptides as a Potential Drug Delivery System for Effective Treatment of Diabetes.

Type 2 diabetes (T2D) is characterized by hyperglycemia resulting from the body's inability to produce and/or use insulin. Patients with T2D often have hyperinsulinemia, dyslipidemia, inflammation, and oxidative stress, which then lead to hypertension, chronic kidney disease, cardiovascular disease, and increased risk of morbidity and mortality (9th leading cause globally). Insulin and related pharmacological therapies are widely used to manage T2D, despite their limitations. Efficient drug delivery systems (DDS) that control drug kinetics may decrease side effects, allow for efficient targeting, and increase the bioavailability of drugs to achieve maximum therapeutic benefits. Thus, the development of effective DDS is crucial to beat diabetes. Here, we introduced a highly bioavailable vector, cell-penetrating peptides (CPPs), as a powerful DDS to overcome limitations of free drug administration. CPPs are short peptides that serve as a potent tool for delivering therapeutic agents across cell membranes. Various cargoes, including proteins, DNA, RNA, liposomes, therapeutic molecules, and nanomaterials, generally retain their bioactivity upon entering cells. The mechanisms of CPPs/cargoes intracellular entry are classified into two parts: endocytic pathways and direct membrane translocation. In this article, we focus on the applications of CPPs/therapeutic agents in the treatment of diabetes. Hypoglycemic drugs with CPPs intervention can enhance therapeutic effectiveness, and CPP-mediated drug delivery can facilitate the actions of insulin. Numerous studies indicate that CPPs can effectively deliver insulin, produce synergistic effects with immunosuppressants for successful pancreatic islet xenotransplantation, prolong pharmacokinetics, and retard diabetic nephropathy. We suggest that CPPs can be a new generation of drug delivery systems for effective treatment and management of diabetes and diabetes-associated complications.

Improved production of GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pigs for xenotransplantation by recloning.

Multiple genetic modification is necessary for successful xenotransplantation from pigs. However, multiple-genetically modified cells usually suffer from various drug selections and long-term in vitro culture, which have a poor performance for somatic cell nuclear transfer (SCNT) to produce genetically modified pigs. We used to generate GTKO/hCD55/hCD59 triple-gene modified pigs by using drug-selective cell lines for SCNT, but the majority of cloned pigs were transgenic-negative individuals. In this study, to improve the production efficiency of multiple genetically modified pigs, we performed the recloning process by using transgenic porcine fetal fibroblast cells. As a result, two fetuses expressing hCD55 and hCD59 were obtained from 12 live-cloned fetuses, and one carrying high transgene expression was selected as a source of donor cells for recloning. Then we obtained 12 cloned piglets, all GTKO and carrying hCD55 and hCD59. Both hCD55 and hCD59 were expressed in fibroblast cells, but the expression levels of hCD55 and hCD59 were different among these piglets. Furthermore, piglet P5# had the highest expression of hCD55 and hCD59 in fibroblast cells than other piglets. Correspondingly, fibroblast cells of piglet P5# had significantly higher resistance against human serum-mediated cytolysis than those of piglet P11#. In conclusion, our results firstly provide support for improving efficiency of generating multiple genetically modified pig by recloning.

Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice

BackgroundXenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis.MethodsSSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L−1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis.ResultsSupplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice.ConclusionsIn conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.

Toward a solution for cardiac failure in the newborn.

The newborn infant with severe cardiac failure owed to congenital structural heart disease or cardiomyopathy poses a daunting therapeutic challenge. The ideal solution for both might be cardiac transplantation if availability of hearts was not limiting and if tolerance could be induced, obviating toxicity of immunosuppressive therapy. If one could safely and effectively exploit neonatal tolerance for successful xenotransplantation of the heart, the challenge of severe cardiac failure in the newborn infant might be met. We discuss the need, the potential for applying neonatal tolerance in the setting of xenotransplantation and the possibility that other approaches to this problem might emerge.