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The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of pluripotent stem cells in vitro . To explore the ontogeny of human and primate pluripotency, here we perform comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys ( Macaca fascicularis ). We show that after specification in the blastocysts, EPI from cynomolgus monkeys (cyEPI) undergoes major transcriptome changes on implantation. Thereafter, while generating gastrulating cells, cyEPI stably maintains its transcriptome over a week, retains a unique set of pluripotency genes and acquires properties for ‘neuron differentiation’. Human and monkey pluripotent stem cells show the highest similarity to post-implantation late cyEPI, which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI and epiblast-like cells in vitro . These findings not only reveal the divergence and coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro . Using a single-cell sequencing analysis in monkey embryos, and comparing the genes expressed during early development in this species with those in mice and in human pluripotent stem cells, the authors define characteristics of pluripotency ontogeny across mammalian species. Species differences in developing pluripotent stem cells Using a single-cell-sequencing-based analysis in monkey embryos, and comparing the genes expressed during early development in this species and what is known from mouse and human studies, Mitinori Saitou and colleagues define characteristics of pluripotency ontogeny across mammalian species. They show that, surprisingly, monkey cells undergoing neuronal differentiation continue to express genes associated with pluripotency during gastrulation. The analysis also provides insights into the comparative properties of developmental-stage pluripotent stem cells in key species that will help to establish a basis for better regulation of human pluripotency in vitro .

Embryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation non-human primate embryos, we uncover that ISL1 , a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development. Human and murine embryonic development has disparities, highlighting the need for primate systems. Here, the authors construct a post-implantation transcriptional atlas from non-human primate embryos and show ISL1 controls a gene regulatory network in the amnion required for mesoderm formation.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response 1 . Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters ( Mesocricetus auratus ), mice ( Mus musculus ) and cynomolgus macaques ( Macaca fascicularis ), and—concomitantly—protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model 2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate. A candidate vaccine against SARS-CoV-2 that uses the yellow fever 17D live-virus vector is highly efficacious and displays a favourable safety profile in Syrian hamster, mouse and cynomolgus macaque models.

Gene-editing technologies, which include the CRISPR–Cas nucleases 1 – 3 and CRISPR base editors 4 , 5 , have the potential to permanently modify disease-causing genes in patients 6 . The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys ( Macaca fascicularis ). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a ‘once-and-done’ approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide 7 ), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs. In a cynomolgus macaque model, CRISPR base editors delivered in lipid nanoparticles are shown to efficiently and stably knock down PCSK9 in the liver to reduce levels of PCSK9 and low-density lipoprotein cholesterol in the blood.

Animal models are an integral part of the drug development and evaluation process. However, they are unsurprisingly imperfect reflections of humans, and the extent and nature of many immunological differences are unknown. With the rise of targeted and biological therapeutics, it is increasingly important that we understand the molecular differences in the immunological behavior of humans and model organisms. However, very few antibodies are raised against non-human primate antigens, and databases of cross-reactivity between species are incomplete. Thus, we screened 332 antibodies in five immune cell populations in blood from humans and four non-human primate species generating a comprehensive cross-reactivity catalog that includes cell type-specificity. We used this catalog to create large mass cytometry universal cross-species phenotyping and signaling panels for humans, along with three of the model organisms most similar to humans: rhesus and cynomolgus macaques and African green monkeys; and one of the mammalian models most widely used in drug development: C57BL/6 mice. As a proof-of-principle, we measured immune cell signaling responses across all five species to an array of 15 stimuli using mass cytometry. We found numerous instances of different cellular phenotypes and immune signaling events occurring within and between species, and detailed three examples (double-positive T cell frequency and signaling; granulocyte response to Bacillus anthracis antigen; and B cell subsets). We also explore the correlation of herpes simian B virus serostatus on the immune profile. Antibody panels and the full dataset generated are available online as a resource to enable future studies comparing immune responses across species during the evaluation of therapeutics.

Studying tissue composition and function in non-human primates (NHPs) is crucial to understand the nature of our own species. Here we present a large-scale cell transcriptomic atlas that encompasses over 1 million cells from 45 tissues of the adult NHP Macaca fascicularis . This dataset provides a vast annotated resource to study a species phylogenetically close to humans. To demonstrate the utility of the atlas, we have reconstructed the cell–cell interaction networks that drive Wnt signalling across the body, mapped the distribution of receptors and co-receptors for viruses causing human infectious diseases, and intersected our data with human genetic disease orthologues to establish potential clinical associations. Our M .  fascicularis cell atlas constitutes an essential reference for future studies in humans and NHPs. A large-scale single-cell transcriptomic atlas of the non-human primate Macaca fascicularis encompasses over 1 million cells from 45 adult tissues.

Few animals have the cognitive faculties or prehensile abilities needed to eliminate tooth-damaging grit from food surfaces. Some populations of monkeys wash sand from foods when standing water is readily accessible, but this propensity varies within groups for reasons unknown. Spontaneous food-washing emerged recently in a group of long-tailed macaques ( Macaca fascicularis ) inhabiting Koram Island, Thailand, and it motivated us to explore the factors that drive individual variability. We measured the mineral and physical properties of contaminant sands and conducted a field experiment, eliciting 1282 food-handling bouts by 42 monkeys. Our results verify two long-standing presumptions: monkeys have a strong aversion to sand and removing it is intentional. Reinforcing this result, we found that monkeys clean foods beyond the point of diminishing returns, a suboptimal behavior that varied with social rank. Dominant monkeys abstained from washing, a choice consistent with the impulses of dominant monkeys elsewhere: to prioritize rapid food intake and greater reproductive fitness over the long-term benefits of prolonging tooth function. While picnics at the beach sound fun, sand is notorious for sticking to food surfaces and nobody likes the feeling of grit on their teeth. Therefore, it is hardly surprising that monkeys living near beaches tend to clean sand from their food. While some briefly brush food with their hands, others wash food items in the ocean with care. Although washing food in water might seem like the best way to eliminate sand, the most dominant monkey in a social group almost never does it. This difference in cleaning behavior raises the possibility that these monkeys make a shrewd calculation in their minds: is eating more quickly worth the risk of tooth damage from sand? To explore this idea, Rosien et al. studied wild monkeys living on Koram Island in Thailand, which are known to wash their food in water. Measuring the properties of the sand that stuck to the food showed that 78% of it is made up of quartz – a mineral that is known to damage teeth. The researchers then studied how much time the monkeys spent washing or brushing slices of cucumber with different amounts of sand on them. This revealed that monkeys spent more time cleaning food with more sand on it, confirming the idea that they are averse to sand on their food and intentionally remove it. The experiments also showed that monkeys tend to spend more time cleaning sand from their food than Rosien et al. had predicted to be necessary, indicating they prioritize this careful cleaning over efficient energy intake. However, this was not the case for the most dominant monkeys in the social group. They favored the quicker but less effective method of brushing food with their hands, suggesting that their main priority is immediate feeding, despite the long-term risk of tooth damage from sand. The findings will be of interest to evolutionary biologists focused on the tradeoffs between foraging behavior and other vital needs, such as growth, reproduction, and ageing. The experiments also suggest that tooth wear can vary among individuals as a result of different cleaning behaviors, rather than just food types, which will be relevant for future studies by paleoanthropologists.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and life. A useful pathological animal model accurately reflecting human pathology is needed to overcome the COVID-19 crisis. In the present study, COVID-19 cynomolgus monkey models including monkeys with underlying diseases causing severe pathogenicity such as metabolic disease and elderly monkeys were examined. Cynomolgus macaques with various clinical conditions were intranasally and/or intratracheally inoculated with SARS-CoV-2. Infection with SARS-CoV-2 was found in mucosal swab samples, and a higher level and longer period of viral RNA was detected in elderly monkeys than in young monkeys. Pneumonia was confirmed in all of the monkeys by computed tomography images. When monkeys were readministrated SARS-CoV-2 at 56 d or later after initial infection all of the animals showed inflammatory responses without virus detection in swab samples. Surprisingly, in elderly monkeys reinfection showed transient severe pneumonia with increased levels of various serum cytokines and chemokines compared with those in primary infection. The results of this study indicated that the COVID-19 cynomolgus monkey model reflects the pathophysiology of humans and would be useful for elucidating the pathophysiology and developing therapeutic agents and vaccines.

Mutation or disruption of the SH3 and ankyrin repeat domains 3 ( SHANK3 ) gene represents a highly penetrant, monogenic risk factor for autism spectrum disorder, and is a cause of Phelan–McDermid syndrome. Recent advances in gene editing have enabled the creation of genetically engineered non-human-primate models, which might better approximate the behavioural and neural phenotypes of autism spectrum disorder than do rodent models, and may lead to more effective treatments. Here we report CRISPR–Cas9-mediated generation of germline-transmissible mutations of SHANK3 in cynomolgus macaques ( Macaca fascicularis ) and their F1 offspring. Genotyping of somatic cells as well as brain biopsies confirmed mutations in the SHANK3 gene and reduced levels of SHANK3 protein in these macaques. Analysis of data from functional magnetic resonance imaging revealed altered local and global connectivity patterns that were indicative of circuit abnormalities. The founder mutants exhibited sleep disturbances, motor deficits and increased repetitive behaviours, as well as social and learning impairments. Together, these results parallel some aspects of the dysfunctions in the SHANK3 gene and circuits, as well as the behavioural phenotypes, that characterize autism spectrum disorder and Phelan–McDermid syndrome. The CRISPR–Cas9-mediated generation of germline-transmissible mutations of SHANK3 in cynomolgus macaques ( Macaca fascicularis ) forms the basis of a non-human-primate model of autism spectrum disorder and Phelan–McDermid syndrome.