Live birth of chimeric monkey with high contribution from embryonic stem cells.

A formal demonstration that mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency is the generation of chimeric animals through early embryo complementation with homologous cells. Whereas such naive pluripotency has been well demonstrated in rodents, poor chimerism has been achieved in other species including non-human primates due to the inability of the donor cells to match the developmental state of the host embryos. Here, we have systematically tested various culture conditions for establishing monkey naive embryonic stem cells and optimized the procedures for chimeric embryo culture. This approach generated an aborted fetus and a live chimeric monkey with high donor cell contribution. A stringent characterization pipeline demonstrated that donor cells efficiently (up to 90%) incorporated into various tissues (including the gonads and placenta) of the chimeric monkeys. Our results have major implications for the study of primate naive pluripotency and genetic engineering of non-human primates.

Base-edited cynomolgus monkeys mimic core symptoms of STXBP1 encephalopathy.

Presynaptic syntaxin binding protein 1 (STXBP1) is essential for neurotransmitter release. Heterozygous mutations in this protein cause STXBP1 encephalopathy (STXBP1-E), which is characterized by intellectual disabilities and epilepsies. Since nonhuman primates closely resemble humans, monkey models may advance studies on the pathogenesis and therapeutic treatments of STXBP1-E. We generated cynomolgus monkeys carrying STXBP1 (R292H) mutation through base editing of in vitro fertilized embryos to mimic a clinical condition. The newborn STXBP1-edited monkeys exhibited focal epilepsy, and the animal that survived beyond the first week postpartum presented typical EEG phenotypes. Biochemical analysis of brain biopsy samples showed reduced levels of STXBP1 (MUNC18-1) and SNARE complex proteins. Single-cell sequencing identified one specific cell cluster that may contribute to encephalopathy. Thus, our case report shows that base-edited STXBP1 mutant monkeys are a good animal model for STXBP1-E, and that a base-editing approach is useful for generating primate models of human genetic disorders.

Characterizing the rhythmic oscillations of gut bacterial and fungal communities and their rhythmic interactions in male cynomolgus monkeys.

The circadian oscillation of gut microbiota plays vital roles in the normal physiology and health of the host. Although the diurnal oscillation of intestinal bacteria has been extensively studied, little relevant work has been done on intestinal fungi. Besides, the rhythmic correlations between bacterial and fungal microbes are also scarcely reported. Here, we investigated the diurnal oscillations of bacterial and fungal communities in male cynomolgus monkeys by performing 16S rRNA and ITS amplicon sequencing. As for bacterial genera, we found that the relative abundance of Prevotella, norank_f_Eubacterium_coprostanoligenes_group, and Peptococcus underwent significant changes at ZT12 (19:00) and exhibited obvious rhythmic oscillations. Consequently, most of the bacterial functions varied at ZT12 and were positively correlated with the bacterial genera norank_f_Eubacterium_coprostanoligenes_group and Prevotella. Among the fungal genera, the relative abundance of Aspergillus and Talaromyces decreased at ZT18 (1:00) and showed slight rhythmic oscillations. As for the fungal function, the undefined saprotroph showed slight rhythmic oscillation and was positively correlated with the fungal genus Aspergillus. Notably, we characterized the correlations between intestinal bacteria and fungi every 6 h over the course of a day and found that the bacterial and fungal microbes interacted closely, with the most bacteria-fungi interactions occurring at ZT12. Our study contributed to a more comprehensive understanding of the diurnal oscillation patterns of bacterial and fungal microbes in male cynomolgus monkeys and uncovered their correlations during a diurnal cycle. IMPORTANCE: The rhythmic oscillation of gut microbiota can impact the physiology activity and disease susceptibility of the host. Until now, most of the studies are focused on bacterial microbes, ignoring other components of gut microbes, such as fungal microbes (mycobiota). Besides, only few studies have addressed the rhythmic correlations between gut bacteria and fungi. Here, we analyzed the rhythmic oscillations of bacterial and fungal communities in male cynomolgus monkeys by performing 16S rRNA and ITS amplicon sequencing. Apart from identifying the rhythmically oscillated bacterial and fungal microbes, we conducted the correlation analysis between these two microbial communities and found that the intestinal bacteria and fungi exhibited close interactions rhythmically, with the most interactions occurring at ZT12. Thus, our study not only investigated the rhythmic oscillations of gut bacterial and fungal communities in male cynomolgus monkeys but also uncovered their rhythmic interactions.

Comparative genomics of macaques and integrated insights into genetic variation and population history.

The crab-eating macaques ( Macaca fascicularis ) and rhesus macaques ( M. mulatta ) are widely studied nonhuman primates in biomedical and evolutionary research. Despite their significance, the current understanding of the complex genomic structure in macaques and the differences between species requires substantial improvement. Here, we present a complete genome assembly of a crab-eating macaque and 20 haplotype-resolved macaque assemblies to investigate the complex regions and major genomic differences between species. Segmental duplication in macaques is ∼42% lower, while centromeres are ∼3.7 times longer than those in humans. The characterization of ∼2 Mbp fixed genetic variants and ∼240 Mbp complex loci highlights potential associations with metabolic differences between the two macaque species (e.g., CYP2C76 and EHBP1L1 ). Additionally, hundreds of alternative splicing differences show post-transcriptional regulation divergence between these two species (e.g., PNPO ). We also characterize 91 large-scale genomic differences between macaques and humans at a single-base-pair resolution and highlight their impact on gene regulation in primate evolution (e.g., FOLH1 and PIEZO2 ). Finally, population genetics recapitulates macaque speciation and selective sweeps, highlighting potential genetic basis of reproduction and tail phenotype differences (e.g., STAB1 , SEMA3F , and HOXD13 ). In summary, the integrated analysis of genetic variation and population genetics in macaques greatly enhances our comprehension of lineage-specific phenotypes, adaptation, and primate evolution, thereby improving their biomedical applications in human diseases.

Nuclear transfer improves the developmental potential of embryos derived from cytoplasmic deficient oocytes.

Embryo development after fertilization is largely determined by the oocyte quality, which is in turn dependent on the competence of both the cytoplasm and nucleus. Here, to improve the efficiency of embryo development from developmentally incompetent oocytes, we performed spindle-chromosome complex transfer (ST) between in vitro matured (IVM) and in vivo matured (IVO) oocytes of the non-human primate rhesus monkey. We observed that the blastocyst rate of embryos derived from transferring the spindle-chromosome complex (SCC) of IVM oocytes into enucleated IVO oocytes was comparable with that of embryos derived from IVO oocytes. After transferring the reconstructed embryos into the uterus of surrogate mothers, two live rhesus monkeys were obtained, indicating that the nuclei of IVM oocytes support both the pre-and post-implantation embryo development of non-human primates.

Characterizing the Pathogenicity and Immunogenicity of Simian Retrovirus Subtype 8 (SRV-8) Using SRV-8-Infected Cynomolgus Monkeys.

Simian retrovirus subtype 8 (SRV-8) infections have been reported in cynomolgus monkeys (Macaca fascicularis) in China and America, but its pathogenicity and immunogenicity are rarely reported. In this work, the SRV-8-infected monkeys were identified from the monkeys with anemia, weight loss, and diarrhea. To clarify the impact of SRV-8 infection on cynomolgus monkeys, infected monkeys were divided into five groups according to disease progression. Hematoxylin (HE) staining and viral loads analysis showed that SRV-8 mainly persisted in the intestine and spleen, causing tissue damage. Additionally, the dynamic variations of blood routine indexes, innate and adaptive immunity, and the transcriptomic changes in peripheral blood cells were analyzed during SRV-8 infection. Compared to uninfected animals, red blood cells, hemoglobin, and white blood cells were reduced in SRV-8-infected monkeys. The percentage of immune cell populations was changed after SRV-8 infection. Furthermore, the number of hematopoietic stem cells decreased significantly during the early stages of SRV-8 infection, and returned to normal levels after antibody-mediated viral clearance. Finally, global transcriptomic analysis in PBMCs from SRV-8-infected monkeys revealed distinct gene expression profiles across different disease stages. In summary, SRV-8 infection can cause severe pathogenicity and immune disturbance in cynomolgus monkeys, and it might be responsible for fatal virus-associated immunosuppressive syndrome.

Cynomolgus-rhesus hybrid macaques serve as a platform for imprinting studies.

Genomic imprinting can lead to allele-specific expression (ASE), where one allele is preferentially expressed more than the other. Perturbations in genomic imprinting or ASE genes have been widely observed across various neurological disorders, notably autism spectrum disorder (ASD). In this study, we crossed rhesus cynomolgus monkeys to produce hybrid monkeys and established a framework to evaluate their allele-specific gene expression patterns using the parental genomes as a reference. Our proof-of-concept analysis of the hybrid monkeys identified 353 genes with allele-biased expression in the brain, enabling us to determine the chromosomal locations of ASE clusters. Importantly, we confirmed a significant enrichment of ASE genes associated with neuropsychiatric disorders, including ASD, highlighting the potential of hybrid monkey models in advancing our understanding of genomic imprinting.

Cynomolgus monkey embryo model captures gastrulation and early pregnancy.

Human stem cell-derived blastoids display similar morphology and cell lineages to normal blastocysts. However, the ability to investigate their developmental potential is limited. Here, we construct cynomolgus monkey blastoids resembling blastocysts in morphology and transcriptomics using naive ESCs. These blastoids develop to embryonic disk with the structures of yolk sac, chorionic cavity, amnion cavity, primitive streak, and connecting stalk along the rostral-caudal axis through prolonged in vitro culture (IVC). Primordial germ cells, gastrulating cells, visceral endoderm/yolk sac endoderm, three germ layers, and hemato-endothelial progenitors in IVC cynomolgus monkey blastoids were observed by single-cell transcriptomics or immunostaining. Moreover, transferring cynomolgus monkey blastoids to surrogates achieves pregnancies, as indicated by progesterone levels and presence of early gestation sacs. Our results reveal the capacity of in vitro gastrulation and in vivo early pregnancy of cynomolgus monkey blastoids, providing a useful system to dissect primate embryonic development without the same ethical concerns and access challenges in human embryo study.

The microRNA cluster miR-30b/-30d prevents tumor cell switch from an epithelial to a mesenchymal-like phenotype in GBC.

As a malignancy of the gastrointestinal tract, gallbladder cancer (GBC) continues to exhibit notable rates of mortality. The current study aimed at investigating the effects associated with miR-30b and miR-30d (miR-30b/-30d) patterns in tumor cells undergoing epithelial-to-mesenchymal transition (EMT) in GBC. It identified that miR-30b and miR-30d, composed as a miRNA cluster, exhibited lower levels in the cancerous tissues from 50 patients with GBC relative to the gallbladder tissues from 35 patients with chronic cholecystitis. As expected, elevated expression of miR-30b/-30d was found to inhibit the EMT process, as evidenced by enhanced E-cadherin and reduced N-cadherin and vimentin in human GBC cells treated with miR-30b mimic, miR-30d mimic, and miR-30b/-30d mimic. Semaphorin-6B (SEMA6B) was identified as a target gene of miR-30b/-30d. Silencing of SEMA6B by its specific small interfering RNA (siRNA) mimicked the effect of miR-30b/-30d upregulation on the GBC cell EMT. Consistently, SEMA6B overexpression promoted this phenotypic switch even in the presence of miR-30b/-30d mimic. The tumorigenicity assay data obtained from nude mice also further supported the notion that miR-30b/-30d inhibited EMT of GBC cells. Thus, based on the key findings of the current study, we concluded that the miR-30b/-30d cluster may provide a potential avenue for targeting mesenchymal-like, invasive tumor cells in GBC.

Cloning of a gene-edited macaque monkey by somatic cell nuclear transfer.

Cloning of macaque monkeys by somatic cell nucleus transfer (SCNT) allows the generation of monkeys with uniform genetic backgrounds that are useful for the development of non-human primate models of human diseases. Here, we report the feasibility of this approach by SCNT of fibroblasts from a macaque monkey (Macaca fascicularis), in which a core circadian transcription factor BMAL1 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing (see accompanying paper). Out of 325 SCNT embryos transferred into 65 surrogate monkeys, we cloned five macaque monkeys with BMAL1 mutations in both alleles without mosaicism, with nuclear genes identical to that of the fibroblast donor monkey. Further peripheral blood mRNA analysis confirmed the complete absence of the wild-type BMAL1 transcript. This study demonstrates that the SCNT approach could be used to generate cloned monkeys from fibroblasts of a young adult monkeys and paves the way for the development of macaque monkey disease models with uniform genetic backgrounds.