The α-Gal KO Mouse Animal Model is a Reliable and Predictive Tool for the Immune-Mediated Calcification Assessment of Heart Valve Bioprostheses.

BACKGROUND: Recent studies highlighted the presence of anti-α-Gal antibodies in patients implanted with commercial bioprosthetic heart valves (BHVs). BHVs expose residual α-Gal xenoantigen and their recognition by the circulating anti-Gal antibodies leads to opsonization of the device's tissue component with the consequent triggering of a deterioration pathway that culminates with calcification. Small animal models such as mice and rats have been broadly involved in the in vivo testing of biomaterials by subcutaneous implantation, especially for the effectiveness of BHVs anti-calcific treatments. However, since models employed for this purpose express α-Gal antigen, the implantation of BHVs' leaflets does not elicit a proper immunological response, so the calcification propensity may be dramatically underestimated. METHODS: An α-Gal knockout (KO) mouse model has been created, using the CRISP/Cas9 approach, and adopted to assess the calcification potential of commercial BHVs leaflets through the surgical implantation in the back subcutis area. Calcium quantification was performed by inductively coupled plasma analysis; immune response against the BHVs leaflets and α-Gal silencing was evaluated through immunological assays. RESULTS: Two months after the implantation of commercial BHV leaflets, the anti-Gal antibody titers in KO mice doubled when compared with those found in wild-type (WT) ones. Leaflets explanted from KO mice, after one month, showed a four-time increased calcium deposition concerning the ones explanted from WT. The degree of silencing of α-Gal varied, depending on the specific organ that was assessed. In any case, the animal model was suitable for evaluating implanted tissue responses. CONCLUSIONS: Such mouse model proved to be an accurate tool for the study of the calcific propensity of commercial BHVs leaflets than those hitherto used. Given its reliability, it could also be successfully used to study even other diseases in which the possible involvement of α-Gal has been observed.

First-in-human Study With LIS1, a Next-generation Porcine Low Immunogenicity Antilymphocyte Immunoglobulin in Kidney Transplantation.

BACKGROUND: Polyclonal rabbit antithymocyte globulins (ATGs) are commonly used in organ transplantation as induction. Anti-N-glycolylneuraminic acid carbohydrate antibodies which develop in response to rabbit carbohydrate antigens might lead to unwanted systemic inflammation. LIS1, the first new generation of antilymphocyte globulins (ALGs) derived from double knockout swine, lacking carbohydrate xenoantigens was already tested in nonhuman primates and rodent models. METHODS: This open-label, single-site, dose escalation, first-in-human, phase 1 study evaluated the safety, T cell depletion, pharmacokinetics, and pharmacodynamics of LIS1. In an ascending dose cohort (n = 5), a primary kidney transplant recipient at low immunologic risk (panel reactive antibody [PRA] < 20%), received LIS1 for 5 d at either 0.6, 1, 3, 6, or 8 mg/kg. After each patient completed treatment, the data safety monitoring board approved respective dose escalation. In the therapeutic dose cohort (n = 5) in patients with PRA <50% without donor specific antibodies, 2 patients received 8 mg/kg and 3 patients 10 mg/kg. RESULTS: CD3+ T cell depletion <100/mm3 at day 2 was observed in all patients who received 6, 8, and 10 mg/kg of LIS1. The terminal half-life of LIS1 was 33.7 d with linearity in its disposition. Lymphocyte repopulation was fast and pretransplant lymphocyte subpopulation counts recovered within 2-4 wk. LIS1 was well tolerated, neither cytokine release syndrome nor severe thrombocytopenia or leukopenia were noticed. Antibodies to LIS1 were not detected. CONCLUSIONS: In this first-in-human trial, genome-edited swine-derived polyclonal LIS1 ALG was well tolerated, did not elicit antidrug antibodies, and caused time-limited T cell depletion in low- and medium-risk kidney transplant recipients.

Changes in glial cell activation and extracellular vesicles production precede the onset of disease symptoms in transgenic hSOD1G93A pigs.

SOD1 gene is associated with progressive motor neuron degeneration in the familiar forms of amyotrophic lateral sclerosis. Although studies on mutant human SOD1 transgenic rodent models have provided important insights into disease pathogenesis, they have not led to the discovery of early biomarkers or effective therapies in human disease. The recent generation of a transgenic swine model expressing the human pathological hSOD1G93A gene, which recapitulates the course of human disease, represents an interesting tool for the identification of early disease mechanisms and diagnostic biomarkers. Here, we analyze the activation state of CNS cells in transgenic pigs during the disease course and investigate whether changes in neuronal and glial cell activation state can be reflected by the amount of extracellular vesicles they release in biological fluids. To assess the activation state of neural cells, we performed a biochemical characterization of neurons and glial cells in the spinal cords of hSOD1G93A pigs during the disease course. Quantification of EVs of CNS cell origin was performed in cerebrospinal fluid and plasma of transgenic pigs at different disease stages by Western blot and peptide microarray analyses. We report an early activation of oligodendrocytes in hSOD1G93A transgenic tissue followed by astrocyte and microglia activation, especially in animals with motor symptoms. At late asymptomatic stage, EV production from astrocytes and microglia is increased in the cerebrospinal fluid, but not in the plasma, of transgenic pigs reflecting donor cell activation in the spinal cord. Estimation of EV production by biochemical analyses is corroborated by direct quantification of neuron- and microglia-derived EVs in the cerebrospinal fluid by a Membrane Sensing Peptide enabled on-chip analysis that provides fast results and low sample consumption. Collectively, our data indicate that alteration in astrocytic EV production precedes the onset of disease symptoms in the hSODG93A swine model, mirroring donor cell activation in the spinal cord, and suggest that EV measurements from the cells first activated in the ALS pig model, i.e. OPCs, may further improve early disease detection.

In vitro fertilization program in white rhinoceros.

In brief: To save endangered rhinoceros species, assisted reproductive technologies are warranted. We here report in vitro blastocyst generation of the Near-Threatened Southern white rhinoceros and, for the first time, also of the technically Extinct Northern white rhinoceros. Abstract: The Anthropocene is marked by a dramatic biodiversity decline, particularly affecting the family Rhinocerotidae. Three of five extant species are listed as Critically Endangered (Sumatran, Javan, black rhinoceros), one as Vulnerable (Indian rhinoceros), and only one white rhino (WR) subspecies, the Southern white rhinoceros (SWR), after more than a century of successful protection is currently classified as Near Threatened by the IUCN, while numbers again are declining. Conversely, in 2008, the SWR's northern counterpart and second WR subspecies, the Northern white rhinoceros (NWR), was declared extinct in the wild. Safeguarding these vanishing keystone species urgently requires new reproductive strategies. We here assess one such strategy, the novel in vitro fertilization program in SWR and - for the first-time NWR - regarding health effects, donor-related, and procedural factors. Over the past 8 years, we performed 65 procedures in 22 white rhinoceros females (20 SWR and 2 NWR) comprising hormonal ovarian stimulation, ovum pick-up (OPU), in vitro oocyte maturation, fertilization, embryo culture, and blastocyst cryopreservation, at an efficiency of 1.0 ± 1.3 blastocysts per OPU, generating 22 NWR, 19 SWR and 10 SWR/NWR hybrid blastocysts for the future generation of live offspring.

The neonatal southern white rhinoceros ovary contains oogonia in germ cell nests.

The northern white rhinoceros is functionally extinct with only two females left. Establishing methods to culture ovarian tissues, follicles, and oocytes to generate eggs will support conservation efforts using in vitro embryo production. To the best of our knowledge, this is the first description of the structure and molecular signature of any rhinoceros, more specifically, we describe the neonatal and adult southern white rhinoceros (Ceratotherium simum simum) ovary; the closest relation of the northern white rhinoceros. Interestingly, all ovaries contain follicles despite advanced age. Analysis of the neonate reveals a population of cells molecularly characterised as mitotically active, pluripotent with germ cell properties. These results indicate that unusually, the neonatal ovary still contains oogonia in germ cell nests at birth, providing an opportunity for fertility preservation. Therefore, utilising ovaries from stillborn and adult rhinoceros can provide cells for advanced assisted reproductive technologies and investigating the neonatal ovaries of other endangered species is crucial for conservation.

Differential Immune Response to Bioprosthetic Heart Valve Tissues in the α1,3Galactosyltransferase-Knockout Mouse Model.

Structural valve deterioration (SVD) of bioprosthetic heart valves (BHVs) has great clinical and economic consequences. Notably, immunity against BHVs plays a major role in SVD, especially when implanted in young and middle-aged patients. However, the complex pathogenesis of SVD remains to be fully characterized, and analyses of commercial BHVs in standardized-preclinical settings are needed for further advancement. Here, we studied the immune response to commercial BHV tissue of bovine, porcine, and equine origin after subcutaneous implantation into adult α1,3-galactosyltransferase-knockout (Gal KO) mice. The levels of serum anti-galactose α1,3-galactose (Gal) and -non-Gal IgM and IgG antibodies were determined up to 2 months post-implantation. Based on histological analyses, all BHV tissues studied triggered distinct infiltrating cellular immune responses that related to tissue degeneration. Increased anti-Gal antibody levels were found in serum after ATS 3f and Freedom/Solo implantation but not for Crown or Hancock II grafts. Overall, there were no correlations between cellular-immunity scores and post-implantation antibodies, suggesting these are independent factors differentially affecting the outcome of distinct commercial BHVs. These findings provide further insights into the understanding of SVD immunopathogenesis and highlight the need to evaluate immune responses as a confounding factor.

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos.

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts (n = 3) and blastocysts (n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro (n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.

LIS1, a glyco-humanized swine polyclonal anti-lymphocyte globulin, as a novel induction treatment in solid organ transplantation.

Anti-thymocyte or anti-lymphocyte globulins (ATGs/ALGs) are immunosuppressive drugs used in induction therapies to prevent acute rejection in solid organ transplantation. Because animal-derived, ATGs/ALGs contain highly immunogenic carbohydrate xenoantigens eliciting antibodies that are associated with subclinical inflammatory events, possibly impacting long-term graft survival. Their strong and long-lasting lymphodepleting activity also increases the risk for infections. We investigated here the in vitro and in vivo activity of LIS1, a glyco-humanized ALG (GH-ALG) produced in pigs knocked out for the two major xeno-antigens αGal and Neu5Gc. It differs from other ATGs/ALGs by its mechanism of action excluding antibody-dependent cell-mediated cytotoxicity and being restricted to complement-mediated cytotoxicity, phagocyte-mediated cytotoxicity, apoptosis and antigen masking, resulting in profound inhibition of T-cell alloreactivity in mixed leucocyte reactions. Preclinical evaluation in non-human primates showed that GH-ALG dramatically reduced CD4+ (p=0.0005,***), CD8+ effector T cells (p=0.0002,***) or myeloid cells (p=0.0007,***) but not T-reg (p=0.65, ns) or B cells (p=0.65, ns). Compared with rabbit ATG, GH-ALG induced transient depletion (less than one week) of target T cells in the peripheral blood (<100 lymphocytes/L) but was equivalent in preventing allograft rejection in a skin allograft model. The novel therapeutic modality of GH-ALG might present advantages in induction treatment during organ transplantation by shortening the T-cell depletion period while maintaining adequate immunosuppression and reducing immunogenicity.

Application of decision tools to ethical analysis in biodiversity conservation.

Achieving ethically responsible decisions is crucial for the success of biodiversity conservation projects. We adapted the ethical matrix, decision tree, and Bateson's cube to assist in the ethical analysis of complex conservation scenarios by structuring these tools so that they can implement the different value dimensions (environmental, social, and animal welfare) involved in conservation ethics. We then applied them to a case study relative to the decision-making process regarding whether or not to continue collecting biomaterial on the oldest of the two remaining northern white rhinoceroses (Ceratotherium simum cottoni), a functionally extinct subspecies of the white rhinoceros. We used the ethical matrix to gather ethical pros and cons and as a starting point for a participatory approach to ethical decision-making. We used decision trees to compare the different options at stake on the basis of a set of ethical desiderata. We used Bateson's cube to establish a threshold of ethical acceptability and model the results of a simple survey. The application of these tools proved to be pivotal in structuring the decision-making process and in helping reach a shared, reasoned, and transparent decision on the best option from an ethical point of view among those available.

Que se logren decisiones éticamente responsables es crucial para el éxito de los proyectos de conservación de la biodiversidad. Adaptamos la matriz ética, el árbol de decisión y el cubo de Bateson para apoyar con el análisis ético de escenarios de conservación compleja mediante la estructuración de estas herramientas de tal manera que puedan ejecutar las diferentes dimensiones de valor (ambiental, social y bienestar animal) involucradas en la ética de la conservación. Después aplicamos las herramientas a un estudio de caso relacionado con el proceso de toma de decisiones respecto a si se debe seguir o no recolectando material biológico del rinoceronte blanco del norte (Ceratotherium simum cottoni) más viejo (una subespecie funcionalmente extinta) de los dos que existen. Usamos la matriz ética como un punto de partida para una estrategia participativa para la toma ética de decisiones y para recopilar los pros y contras éticos. Usamos el árbol de decisión para comparar las diferentes opciones en juego con base en un conjunto de deseos éticos. Usamos el cubo de Bateson para establecer un umbral de aceptación ética y modelar los resultados de una encuesta simple. La aplicación de estas herramientas demostró ser central en la estructuración del proceso de toma de decisiones y en el apoyo para lograr una decisión compartida, razonada y transparente sobre la mejor opción a partir de un punto de vista ético entre aquellos disponibles.

Unexpected impairment of INa underpins reentrant arrhythmias in a knock-in swine model of Timothy syndrome.

Timothy syndrome 1 (TS1) is a multi-organ form of long QT syndrome associated with life-threatening cardiac arrhythmias, the organ-level dynamics of which remain unclear. In this study, we developed and characterized a novel porcine model of TS1 carrying the causative p.Gly406Arg mutation in CACNA1C, known to impair CaV1.2 channel inactivation. Our model fully recapitulated the human disease with prolonged QT interval and arrhythmic mortality. Electroanatomical mapping revealed the presence of a functional substrate vulnerable to reentry, stemming from an unforeseen constitutional slowing of cardiac activation. This signature substrate of TS1 was reliably identified using the reentry vulnerability index, which, we further demonstrate, can be used as a benchmark for assessing treatment efficacy, as shown by testing of multiple clinical and preclinical anti-arrhythmic compounds. Notably, in vitro experiments showed that TS1 cardiomyocytes display Ca2+ overload and decreased peak INa current, providing a rationale for the arrhythmogenic slowing of impulse propagation in vivo.

Robust induction of primordial germ cells of white rhinoceros on the brink of extinction.

In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.

Induced pluripotent stem cells and cerebral organoids from the critically endangered Sumatran rhinoceros.

Less than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats to the species and lead to a continuous population decline. To stop the erosion of genetic diversity, reintroduction of genetic material is indispensable. However, as the propagation rate of captive breeding is far too low, innovative technologies have to be developed. Induced pluripotent stem cells (iPSCs) are a powerful tool to fight extinction. They give rise to each cell within the body including gametes and provide a unique modality to preserve genetic material across time. Additionally, they enable studying species-specific developmental processes. Here, we generate iPSCs from the last male Malaysian SR Kertam, who died in 2019, and characterize them comprehensively. Differentiation in cells of the three germ layers and cerebral organoids demonstrate their high quality and great potential for supporting the rescue of this critically endangered species.

Oxidative Stress in Structural Valve Deterioration: A Longitudinal Clinical Study.

The cause of structural valve deterioration (SVD) is unclear. Therefore, we investigated oxidative stress markers in sera from patients with bioprosthetic heart valves (BHVs) and their association with SVD. Blood samples were taken from SVD (Phase A) and BHV patients during the first 24 (Phase B1) and >48 months (Phase B2) after BHV implantation to assess total antioxidant capacity (TAC), malondialdehyde (MDA), and nitrotyrosine (NT). The results show that MDA levels increased significantly 1 month after surgery in all groups but were higher at 6 months only in incipient SVD patients. NT levels increased gradually for the first 24 months after implantation in the BHV group. Patients with transcatheter aortic valve implantation (TAVI) showed even higher levels of stress markers. After >48 months, MDA and NT continued to increase in BHV patients with a further elevation after 60-72 months; however, these levels were significantly lower in the incipient and established SVD groups. In conclusion, oxidative stress may play a significant role in SVD, increasing early after BHV implantation, especially in TAVI cases, and also after 48 months' follow-up, but decreasing when SVD develops. Oxidative stress potentially represents a target of therapeutic intervention and a biomarker of BHV dysfunction.

Ethical Analysis of the Application of Assisted Reproduction Technologies in Biodiversity Conservation and the Case of White Rhinoceros (Ceratotherium simum) Ovum Pick-Up Procedures.

Originally applied on domestic and lab animals, assisted reproduction technologies (ARTs) have also found application in conservation breeding programs, where they can make the genetic management of populations more efficient, and increase the number of individuals per generation. However, their application in wildlife conservation opens up new ethical scenarios that have not yet been fully explored. This study presents a frame for the ethical analysis of the application of ART procedures in conservation based on the Ethical Matrix (EM), and discusses a specific case study-ovum pick-up (OPU) procedures performed in the current conservation efforts for the northern white rhinoceros (Ceratotherium simum cottoni)-providing a template for the assessment of ART procedures in projects involving other endangered species.

Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction.

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

Improved efficiencies in the generation of multigene-modified pigs by recloning and using sows as the recipient.

This study was performed to improve production efficiency at the level of recipient pig and donor nuclei of transgenic cloned pigs used for xenotransplantation. To generate transgenic pigs, human endothelial protein C receptor (hEPCR) and human thrombomodulin (hTM) genes were introduced using the F2A expression vector into GalT-/-/hCD55+ porcine neonatal ear fibroblasts used as donor cells and cloned embryos were transferred to the sows and gilts. Cloned fetal kidney cells were also used as donor cells for recloning to increase production efficiency. Pregnancy and parturition rates after embryo transfer and preimplantation developmental competence were compared between cloned embryos derived from adult and fetal cells. Significantly higher parturition rates were shown in the group of sows (50.0 vs. 4.1%), natural oestrus (20.8 vs. 0%), and ovulated ovary (16.7 vs. 5.6%) compared with gilt, induced and non-ovulated, respectively (P < 0.05). When using gilts as recipients, final parturitions occurred in only the fetal cell groups and significantly higher blastocyst rates (15.1% vs. 21.3%) were seen (P < 0.05). Additionally, gene expression levels related to pluripotency were significantly higher in the fetal cell group (P < 0.05). In conclusion, sows can be recommended as recipients due to their higher efficiency in the generation of transgenic cloned pigs and cloned fetal cells also can be recommended as donor cells through correct nuclear reprogramming.

25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Current applications of SCNT in advanced breeding and genome editing in livestock.

SCNT (somatic cell nuclear transfer) has complemented the toolbox of ARTs offering yet another technique to reproduce animals in an unprecedented way. Despite remarkable achievements, SCNT suffers low efficiency, high pregnancy losses and higher than normal stillbirth rates that makes it an expensive technique to reproduce animals. Moreover, due to welfare issues associated with gestation and the newborn offspring, it is banned in some countries. It has become evident that these problems are of epigenetic nature associated with incomplete genome reprogramming, observed more frequently in ruminants and less often and of minor degree in pigs and horses. Genome editing is enormously benefiting from SCNT to turn genome edited cells into animals, even if zygote microinjection of CRISPR/Cas9 will become an alternative route in some occasions. SCNT will also be a route to reprogram somatic cell to pluripotency since bona fide iPSC in livestock are missing while embryonic stem cells have been now established. This opens the way to other technologies like the development of artificial gametes or interspecies nuclear transfer. To strengthen its commercial applications, SCNT will face three major challenges, that is, intellectual property (extremely unclear in genome editing), regulatory approval by the relevant authorities of the resuting potential products and finally, acceptance by the public who will eventually decide with its behavior the life or the death of the technology.

An Ethical Assessment Tool (ETHAS) to Evaluate the Application of Assisted Reproductive Technologies in Mammals' Conservation: The Case of the Northern White Rhinoceros (Ceratotherium simum cottoni).

Assisted reproductive technologies (ARTs) can make a difference in biodiversity conservation. Their application, however, can create risks and raise ethical issues that need addressing. Unfortunately, there is a lack of attention to the topic in the scientific literature and, to our knowledge, there is no tool for the ethical assessment of ARTs in the context of conservation that has been described. This paper reports the first applications of the Ethical Assessment Tool (ETHAS) to trans-rectal ovum pick-up (OPU) and in vitro fertilization (IVF) procedures used in a northern white rhinoceros (Ceratotherium simum cottoni) conservation project. The ETHAS consists of two checklists, the Ethical Evaluation Sheet and the Ethical Risk Assessment, and is specifically customized for each ART procedure. It provides an integrated, multilevel and standardized self-assessment of the procedure under scrutiny, generating an ethical acceptability ranking (totally, partially, not acceptable) and a risk rank (low, medium, high), and, hence, allows for implementing measures to address or manage issues beforehand. The application of the ETHAS to the procedures performed on the northern white rhinoceros was effective in ensuring a high standard of procedures, contributing to the acceptability and improved communication among the project's partners. In turn, the tool itself was also refined through an iterative consultation process between experts and stakeholders.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID-19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrates, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3-galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate-N-acetylneuraminic acid hydroxylase and α1,3-galactosyl-transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain to produce glyco-humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3-galactose epitopes. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized spike/angiotensin converting enzyme-2 interaction at a concentration <1 µg/mL, and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. We also found that pig GH-pAb Fc domains fail to interact with human Fc receptors, thereby avoiding macrophage-dependent exacerbated inflammatory responses and a possible antibody-dependent enhancement. These data and the accumulating safety advantages of using GH-pAbs in humans warrant clinical assessment of XAV-19 against COVID-19.

Artificially produced gametes in mice, humans and other species.

The production of gametes from pluripotent stem cells in culture, also known as invitro gametogenesis, will make an important contribution to reproductive biology and regenerative medicine, both as a unique tool for understanding germ cell development and as an alternative source of gametes for reproduction. Invitro gametogenesis was developed using mouse pluripotent stem cells but is increasingly being applied in other mammalian species, including humans. In principle, the entire process of germ cell development is nearly reconstitutable in culture using mouse pluripotent stem cells, although the fidelity of differentiation processes and the quality of resultant gametes remain to be refined. The methodology in the mouse system is only partially applicable to other species, and thus it must be optimised for each species. In this review, we update the current status of invitro gametogenesis in mice, humans and other animals, and discuss challenges for further development of this technology.