Ex utero monkey embryogenesis from blastocyst to early organogenesis.

The third and fourth weeks of gestation in primates are marked by several developmental milestones, including gastrulation and the formation of organ primordia. However, our understanding of this period is limited due to restricted access to in vivo embryos. To address this gap, we developed an embedded 3D culture system that allows for the extended ex utero culture of cynomolgus monkey embryos for up to 25 days post-fertilization. Morphological, histological, and single-cell RNA-sequencing analyses demonstrate that ex utero cultured monkey embryos largely recapitulated key events of in vivo development. With this platform, we were able to delineate lineage trajectories and genetic programs involved in neural induction, lateral plate mesoderm differentiation, yolk sac hematopoiesis, primitive gut, and primordial germ-cell-like cell development in monkeys. Our embedded 3D culture system provides a robust and reproducible platform for growing monkey embryos from blastocysts to early organogenesis and studying primate embryogenesis ex utero.

Resurrection of endogenous retroviruses during aging reinforces senescence.

Whether and how certain transposable elements with viral origins, such as endogenous retroviruses (ERVs) dormant in our genomes, can become awakened and contribute to the aging process is largely unknown. In human senescent cells, we found that HERVK (HML-2), the most recently integrated human ERVs, are unlocked to transcribe viral genes and produce retrovirus-like particles (RVLPs). These HERVK RVLPs constitute a transmissible message to elicit senescence phenotypes in young cells, which can be blocked by neutralizing antibodies. The activation of ERVs was also observed in organs of aged primates and mice as well as in human tissues and serum from the elderly. Their repression alleviates cellular senescence and tissue degeneration and, to some extent, organismal aging. These findings indicate that the resurrection of ERVs is a hallmark and driving force of cellular senescence and tissue aging.

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo.

Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.

Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys.

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.

A developmental landscape of 3D-cultured human pre-gastrulation embryos.

Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms1,2 that do not recapitulate the in vivo conditions3-5. Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior-posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis.

nBEST: Deep-learning-based non-human primates Brain Extraction and Segmentation Toolbox across ages, sites and species.

Accurate processing and analysis of non-human primate (NHP) brain magnetic resonance imaging (MRI) serves an indispensable role in understanding brain evolution, development, aging, and diseases. Despite the accumulation of diverse NHP brain MRI datasets at various developmental stages and from various imaging sites/scanners, existing computational tools designed for human MRI typically perform poor on NHP data, due to huge differences in brain sizes, morphologies, and imaging appearances across species, sites, and ages, highlighting the imperative for NHP-specialized MRI processing tools. To address this issue, in this paper, we present a robust, generic, and fully automated computational pipeline, called non-human primates Brain Extraction and Segmentation Toolbox (nBEST), whose main functionality includes brain extraction, non-cerebrum removal, and tissue segmentation. Building on cutting-edge deep learning techniques by employing lifelong learning to flexibly integrate data from diverse NHP populations and innovatively constructing 3D U-NeXt architecture, nBEST can well handle structural NHP brain MR images from multi-species, multi-site, and multi-developmental-stage (from neonates to the elderly). We extensively validated nBEST based on, to our knowledge, the largest assemblage dataset in NHP brain studies, encompassing 1,469 scans with 11 species (e.g., rhesus macaques, cynomolgus macaques, chimpanzees, marmosets, squirrel monkeys, etc.) from 23 independent datasets. Compared to alternative tools, nBEST outperforms in precision, applicability, robustness, comprehensiveness, and generalizability, greatly benefiting downstream longitudinal, cross-sectional, and cross-species quantitative analyses. We have made nBEST an open-source toolbox (https://github.com/TaoZhong11/nBEST) and we are committed to its continual refinement through lifelong learning with incoming data to greatly contribute to the research field.

C9orf72 poly(PR) aggregation in nucleus induces ALS/FTD-related neurodegeneration in cynomolgus monkeys.

Poly(PR) is a dipeptide repeat protein comprising proline and arginine residues. It is one of the translational product of expanded G4C2 repeats in the C9orf72 gene, and its accumulation is contributing to the neuropathogenesis of C9orf72-associated amyotrophic lateral sclerosis and/or frontotemporal dementia (C9-ALS/FTD). In this study, we demonstrate that poly(PR) protein alone is sufficient to induce neurodegeneration related to ALS/FTD in cynomolgus monkeys. By delivering poly(PR) via AAV, we observed that the PR proteins were located within the nucleus of infected cells. The expression of (PR)50 protein, consisting of 50 PR repeats, led to increased loss of cortical neurons, cytoplasmic lipofuscin, and gliosis in the brain, as well as demyelination and loss of ChAT positive neurons in the spinal cord of monkeys. While, these pathologies were not observed in monkeys expressing (PR)5, a protein comprising only 5 PR repeats. Furthermore, the (PR)50-expressing monkeys exhibited progressive motor deficits, cognitive impairment, muscle atrophy, and abnormal electromyography (EMG) potentials, which closely resemble clinical symptoms seen in C9-ALS/FTD patients. By longitudinally tracking these monkeys, we found that changes in cystatin C and chitinase-1 (CHIT1) levels in the cerebrospinal fluid (CSF) corresponded to the phenotypic progression of (PR)50-induced disease. Proteomic analysis revealed that the major clusters of dysregulated proteins were nuclear-localized, and downregulation of the MECP2 protein was implicated in the toxic process of poly(PR). This research indicates that poly(PR) expression alone induces neurodegeneration and core phenotypes associated with C9-ALS/FTD in monkeys, which may provide insights into the mechanisms of disease pathogenesis.

Knowledge, attitudes and practices of patients with chronic pharyngitis toward laryngopharyngeal reflux in Suzhou, China.

BACKGROUND: This study aimed to investigate the knowledge, attitudes and practices (KAP) of patients with chronic pharyngitis in Suzhou, China toward laryngopharyngeal reflux (LPR). METHODS: This cross-sectional study was conducted in patients with chronic pharyngitis in Suzhou, China at the otolaryngology outpatient clinic of the First Affiliated Hospital of Soochow University between November, 2022, and May, 2023. Data was collected through a self-designed online questionnaire encompassing the sociodemographic characteristics and three dimensions of KAP. The questionnaire was administered using SoJump, and data were exported from this platform. Subsequently, statistical analysis, including Structural Equation Modeling, was performed using SPSS 22 software to evaluate the KAP scores. RESULTS: A total of 487 valid questionnaires were collected, with 275 (56.35%) female patients. The mean score of KAP were 4.76 ± 2.93 (possible range: 0-11), 33.10 ± 4.46 (possible range: 8-40), 31.29 ± 6.04 (possible range: 8-40), respectively. Pearson's correlation analysis showed significant positive correlations between knowledge and attitude dimensions (r = 0.413, P < 0.001), knowledge and practice dimensions (r = 0.355, P < 0.001), and attitude and practice dimensions (r = 0.481, P < 0.001). Structural equation modeling revealed that education exhibited positive effect on knowledge (ß = 0.476, P < 0.001) and attitude (ß = 0.600, P < 0.001), and having family history of chronic pharyngitis showed positive effect on knowledge (ß = 0.580, P = 0.047), experienced with reflux symptoms showed positive effect on knowledge (ß = 0.838, P = 0.001) and attitude (ß = 0.631, P = 0.085). Moreover, knowledge showed positive effect on attitude (ß = 0.555, P < 0.001) and practice (ß = 0.351, P < 0.001). Attitude, in turn, showed positive effect on practice (ß = 0.511, P < 0.001). CONCLUSION: Patients with chronic pharyngitis had inadequate knowledge, positive attitudes and suboptimal practices toward LPR. Education, family history of chronic pharyngitis, experienced with reflux symptoms might have effect on their KAP.

Enhanced effect of recombinant adenoviruses co-expression of ING4 and OSM on anti-tumour activity of laryngeal cancer.

The inhibitor of growth family member 4 (ING4) is one of the ING family genes, serves as a repressor of angiogenesis or tumour growth and suppresses loss of contact inhibition. Oncostatin M (OSM) is a multifunctional cytokine that belongs to the interleukin (IL)-6 subfamily with several biological activities. However, the role of recombinant adenoviruses co-expressing ING4 and OSM (Ad-ING4-OSM) in anti-tumour activity of laryngeal cancer has not yet been identified. Recombinant Ad-ING4-OSM was used to evaluate their combined effect on enhanced anti-tumour activity in Hep-2 cells of laryngeal cancer in vivo. Moreover, in vitro function assays of co-expression of Ad-ING4-OSM were performed to explore impact of co-expression of Ad-ING4-OSM on biological phenotype of laryngeal cancer cell line, that is Hep-2 cells. In vitro, Ad-ING4-OSM significantly inhibited the growth, enhanced apoptosis, altered cell cycle with G1 and G2/M phase arrest, and upregulated the expression of P21, P27, P53 and downregulated survivin in laryngeal cancer Hep-2 cells. Furthermore, in vivo functional experiments of co-expressing of Ad-ING4-OSM demonstrated that solid tumours in the nude mouse model were significantly suppressed, and the co-expressing Ad-ING4-OSM showed a significant upregulation expression of P21, P53, Bax and Caspase-3 and a downregulation of Cox-2, Bcl-2 and CD34. This study for the first time demonstrated the clinical value and the role of co-expressing Ad-ING4-OSM in biological function of laryngeal cancer. This work suggested that co-expressing Ad-ING4-OSM might serve as a potential therapeutic target for laryngeal cancer patients.

Longitudinal brain atlases of early developing cynomolgus macaques from birth to 48 months of age.

Longitudinal brain imaging atlases with densely sampled time-points and ancillary anatomical information are of fundamental importance in studying early developmental characteristics of human and non-human primate brains during infancy, which feature extremely dynamic imaging appearance, brain shape and size. However, for non-human primates, which are highly valuable animal models for understanding human brains, the existing brain atlases are mainly developed based on adults or adolescents, denoting a notable lack of temporally densely-sampled atlases covering the dynamic early brain development. To fill this critical gap, in this paper, we construct a comprehensive set of longitudinal brain atlases and associated tissue probability maps (gray matter, white matter, and cerebrospinal fluid) with totally 12 time-points from birth to 4 years of age (i.e., 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36, and 48 months of age) based on 175 longitudinal structural MRI scans from 39 typically-developing cynomolgus macaques, by leveraging state-of-the-art computational techniques tailored for early developing brains. Furthermore, to facilitate region-based analysis using our atlases, we also provide two popular hierarchy parcellations, i.e., cortical hierarchy maps (6 levels) and subcortical hierarchy maps (6 levels), on our longitudinal macaque brain atlases. These early developing atlases, which have the densest time-points during infancy (to the best of our knowledge), will greatly facilitate the studies of macaque brain development.

DIKA-Nets: Domain-invariant knowledge-guided attention networks for brain skull stripping of early developing macaques.

As non-human primates, macaques have a close phylogenetic relationship to human beings and have been proven to be a valuable and widely used animal model in human neuroscience research. Accurate skull stripping (aka. brain extraction) of brain magnetic resonance imaging (MRI) is a crucial prerequisite in neuroimaging analysis of macaques. Most of the current skull stripping methods can achieve satisfactory results for human brains, but when applied to macaque brains, especially during early brain development, the results are often unsatisfactory. In fact, the early dynamic, regionally-heterogeneous development of macaque brains, accompanied by poor and age-related contrast between different anatomical structures, poses significant challenges for accurate skull stripping. To overcome these challenges, we propose a fully-automated framework to effectively fuse the age-specific intensity information and domain-invariant prior knowledge as important guiding information for robust skull stripping of developing macaques from 0 to 36 months of age. Specifically, we generate Signed Distance Map (SDM) and Center of Gravity Distance Map (CGDM) based on the intermediate segmentation results as guidance. Instead of using local convolution, we fuse all information using the Dual Self-Attention Module (DSAM), which can capture global spatial and channel-dependent information of feature maps. To extensively evaluate the performance, we adopt two relatively-large challenging MRI datasets from rhesus macaques and cynomolgus macaques, respectively, with a total of 361 scans from two different scanners with different imaging protocols. We perform cross-validation by using one dataset for training and the other one for testing. Our method outperforms five popular brain extraction tools and three deep-learning-based methods on cross-source MRI datasets without any transfer learning.

Separation and purification of magnoflorine, spinosin, and 6‴-feruloyspinosin from Ziziphi Spinosae Semen by high-speed counter-current chromatography.

In the study, high-speed counter-current chromatography was used for separation and purification of magnoflorine, spinosin, and 6‴-feruloyspinosin from Ziziphi Spinosae Semen. With n-butanol-ethyl acetate-water (2:3:5, v/v) as the optimum solvent system, about 75 mg of magnoflorine, 110 mg of spinosin, and 40 mg of 6‴-feruloyspinosin were isolated from 0.5 g of crude extract of Z. Spinosae Semen, with the purity of 95.7, 97.2, and 96.4%, respectively. The chemical structures were identified by MS and NMR spectroscopy. In addition, the antidepressant activity of the isolated components was evaluated by PC12 cells injury model and chronic unpredictable mild stress depression mouse model. The results showed that high-speed counter-current chromatography could be used to realize the one-time rapid preparation and separation of magnoflorine, spinosin, and 6‴-feruloyspinosin from Z. Spinosae Semen and compatibility of these isolated components has certain antidepressant activity.

No off-target mutations in functional genome regions of a CRISPR/Cas9-generated monkey model of muscular dystrophy.

CRISPR/Cas9 is now widely used in biomedical research and has great potential for clinical applications. However, the safety and efficacy of this gene-editing technique are significant issues. Recent reports on mouse models and human cells have raised concerns that off-target mutations could hamper applying the CRISPR technology in patients. The high similarities of nonhuman primates to humans in genome content and organization, genetic diversity, physiology, and cognitive abilities have made these animals ideal experimental models for understanding human diseases and developing therapeutics. Off-target mutations of CRISPR/Cas9 have been analyzed in previous studies of nonhuman primates, but no report has investigated genome-wide off-target effects in living monkeys. Here, we used rhesus monkeys in which a genetic disorder mimicking Duchenne muscular dystrophy had previously been produced with CRISPR/Cas9. Using whole-genome sequencing to comprehensively assess on- and off-target mutations in these animals, we found that CRISPR/Cas9-based gene editing is active on the expected genomic sites without producing off-target modifications in other functional regions of the genome. These findings suggest that the CRISPR/Cas9 technique could be relatively safe and effective in modeling genetic disease in nonhuman primates and in future therapeutic research of human diseases.

Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases.

BACKGROUND: Non-human primate (NHP) models can closely mimic human physiological functions and are therefore highly valuable in biomedical research. Genome editing is now developing rapidly due to the precision and efficiency offered by engineered site-specific endonuclease-based systems, such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system. It has been demonstrated that these programmable nucleases can introduce genetic changes in embryos from many species including NHPs. In 2014, we reported the first genetic editing of macaques using TALENs and CRISPR/Cas9. Subsequently, we characterized the phenotype of a methyl CpG binding protein 2 (MECP2)-mutant cynomolgus monkey model of Rett syndrome generated using the TALEN approach. These efforts not only accelerated the advance of modeling genetic diseases in NHPs, but also encouraged us to develop specific gene knock-in monkeys. In this study, we assess the possibility of homologous recombination (HR)-mediated gene replacement using TALENs in monkeys, and generate preimplantation embryos carrying an EmGFP fluorescent reporter constructed in the OCT4 gene. RESULT: We assembled a pair of TALENs specific to the first exon of the OCT4 gene and constructed a donor vector consisting of the homology arms cloned from the monkey genome DNA, flanking an EmGFP cassette. Next, we co-injected the TALENs-coding plasmid and donor plasmid into the cytoplasm of 122 zygotes 6-8 h after fertilization. Sequencing and immunofluorescence revealed that the OCT4-EmGFP knock-in allele had been successfully generated by TALENs-mediated HR at an efficiency of 11.3% (7 out of 62) or 11.1% (1 out of 9), respectively, in monkey embryos. CONCLUSION: We have successfully, for the first time, obtained OCT4-EmGFP knock-in monkey embryos via HR mediated by TALENs. Our results suggest that gene targeting through TALEN-assisted HR is a useful approach to introduce precise genetic modification in NHPs.

MicroRNA-1225-5p acts as a tumor-suppressor in laryngeal cancer via targeting CDC14B.

This study aimed to investigate the role of miRNA-1225-5p (miR-1225) in laryngeal carcinoma (LC). We found that the expression of miR-1225 was suppressed in human LC samples, while CDC14B (cell division cycle 14B) expression was reinforced in comparison with surrounding normal tissues. We also demonstrated that enhanced expression of miR-1225 impaired the proliferation and survival of LC cells, and resulted in G1/S cell cycle arrest. In contrast, reduced expression of miR-1225 promoted cell survival. Moreover, miR-1225 resulted in G1/S cell cycle arrest and enhanced cell death. Further, miR-1225 targets CDC14B 3'-UTR and recovery of CDC14B expression counteracted the suppressive influence of miR-1225 on LC cells. Thus, these findings offer insight into the biological and molecular mechanisms behind the development of LC.

CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH.

Mutations in the DAX1 locus cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH), which manifest with primary adrenal insufficiency and incomplete or absent sexual maturation, respectively. The associated defects in spermatogenesis can range from spermatogenic arrest to Sertoli cell only syndrome. Conclusions from Dax1 knockout mouse models provide only limited insight into AHC/HH disease mechanisms, because mouse models exhibit more extensive abnormalities in testicular development, including disorganized and incompletely formed testis cords with decreased number of peritubular myoid cells and male-to-female sex reversal. We previously reported successful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome targeting in cynomolgus monkeys. Here, we describe a male fetal monkey in which targeted genome editing using CRISPR/Cas9 produced Dax1-null mutations in most somatic tissues and in the gonads. This DAX1-deficient monkey displayed defects in adrenal gland development and abnormal testis architecture with small cords, expanded blood vessels and extensive fibrosis. Sertoli cell formation was not affected. This phenotype strongly resembles findings in human patients with AHC-HH caused by mutations in DAX1. We further detected upregulation of Wnt/ß-catenin-VEGF signaling in the fetal Dax1-deficient testis, suggesting abnormal activation of signaling pathways in the absence of DAX1 as one mechanism of AHC-HH. Our study reveals novel insight into the role of DAX1 in HH and provides proof-of-principle for the generation of monkey models of human disease via CRISPR/Cas9-mediated gene targeting.

Early Parkinson's disease symptoms in α-synuclein transgenic monkeys.

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that can be caused by genetic mutations in α-synuclein (α-syn) or duplication of wild-type α-syn; PD is characterized by the deposition of α-syn aggregates, indicating a gain of toxicity from accumulation of α-syn. Although the major neuropathologic feature of PD is the degeneration of dopaminergic (DA) neurons in the substantia nigra, non-motor symptoms including anxiety, cognitive defect and sleep disorder precede the onset of motor impairment, and many clinical symptoms of PD are not caused by degeneration of DA neurons. Non-human primate models of PD are important for revealing the early pathology in PD and identifying effective treatments. We established transgenic PD rhesus monkeys that express mutant α-syn (A53T). Six transgenic A53T monkeys were produced via lentiviral vector expressing A53T in fertilized monkey eggs and subsequent embryo transfer to surrogates. Transgenic A53T is expressed in the monkey brain and causes age-dependent non-motor symptoms, including cognitive defects and anxiety phenotype, without detectable sleeping disorders. The transgenic α-syn monkeys demonstrate the specific early symptoms caused by mutant α-syn and provide insight into treatment of early PD.