Fetal Monkey Research Articles

Multi-omics identification of extracellular components of the fetal monkey and human neocortex.

During development, precursor cells are continuously and intimately interacting with their extracellular environment, which guides their ability to generate functional tissues and organs. Much is known about the development of the neocortex in mammals. This information has largely been derived from histological analyses, heterochronic cell transplants, and genetic manipulations in mice, and to a lesser extent from transcriptomic and histological analyses in humans. However, these approaches have not led to a characterization of the extracellular composition of the developing neocortex in any species. Here, using a combination of single-cell transcriptomic analyses from published datasets, and our proteomics and immunohistofluorescence analyses, we provide a more comprehensive and unbiased picture of the early developing fetal neocortex in humans and non-human primates. Our findings provide a starting point for further hypothesis-driven studies on structural and signaling components in the developing cortex that had previously not been identified.

Application of ultrasonographic human estimated foetal weight formulas to cynomolgus monkeys (Macaca fascicularis) at 129-132 days of gestation: A comparative study of estimated and actual birthweight.

Cynomolgus monkeys (Macaca fascicularis) are essential in biomedical research, including reproductive studies. However, the application of human estimated foetal weight (EFW) formulas using ultrasonography (USG) in these non-human primates is not well established. This study aims to evaluate the applicability of human EFW formulas for estimating foetal weight in cynomolgus monkeys at approximately 130 days of gestation. Our study involved nine pregnant cynomolgus monkeys. We measured foetal parameters, including biparietal diameter, head circumference, abdominal circumference and femur length using USG. The EFW was calculated using 11 human EFW formulas. The actual birthweight (ABW) was recorded following Cesarean section, the day after the EFW calculation. For comparing EFW and ABW, we employed statistical methods such as mean absolute percentage error (APE) and Bland-Altman analysis. The ABW ranged between 200.36 and 291.33g. Among the 11 formulas, the Combs formula showed the lowest APE (4.3%) and highest correlation with ABW (p<0.001). Notably, EFW and ABW differences for the Combs formula were ≤5% in 66.7% and ≤10% in 100% of cases. The Bland-Altman analysis supported these results, showing that all cases fell within the limits of agreement. The Combs formula is applicable for estimating the weight of cynomolgus monkey fetuses with USG at approximately 130 days of gestation. Our observations suggest that the Combs formula can be applied in the prenatal care and biomedical research of this species.

Mathematical Modeling of Rhesus Cytomegalovirus Transplacental Transmission in Seronegative Rhesus Macaques.

Approximately 0.7% of infants are born with congenital cytomegalovirus (CMV), making it the most common congenital infection. About 1 in 5 congenitally infected babies will suffer long-term sequelae, including sensorineural deafness, intellectual disability, and epilepsy. CMV infection is highly species-dependent, and the rhesus CMV (RhCMV) infection of rhesus monkey fetuses is the only animal model that replicates essential features of congenital CMV (cCMV) infection in humans, including placental transmission, fetal disease, and fetal loss. Using experimental data from RhCMV seronegative rhesus macaques inoculated with RhCMV in the late first to early second trimesters of pregnancy, we built and calibrated a mathematical model for the placental transmission of CMV. The model was then used to study the effect of the timing of inoculation, maternal immune suppression, and hyper-immune globulin infusion on the risk of placental transmission in the context of primary and reactivated chronic maternal CMV infection.

Prenatal Zika virus exposure is associated with lateral geniculate nucleus abnormalities in juvenile rhesus macaques.

Zika virus' neural tropism causes significant neural pathology, particularly in developing fetuses. One of the consistent findings from humans and animal models is that prenatal exposure to Zika virus (ZIKV) causes pathology in the eyes and visual pathways of the brain, although the extent to which this pathology persists over development is not clear. In the present report, we build upon our previous work which demonstrated that full-term rhesus monkey ( Macaca mulatta ) fetuses who were exposed to ZIKV early in gestation had significant pathological abnormalities to the organization of the lateral geniculate nucleus (LGN), a major hub of the visual network. The objective of the present work was to replicate those LGN findings and determine whether such pathology persisted across childhood development. We carried out histological analyses of the LGNs of two juvenile rhesus monkeys who were prenatally exposed to ZIKV and two age-matched controls. Pregnant rhesus monkeys were infected with ZIKV via the intravenous and intra-amniotic routes and tracked across development. Following sacrifice and perfusion, brains were subjected to quantitative neuroanatomical analyses with a focus on the size and structure of the LGN and its composite layers. Early fetal ZIKV exposure resulted in developmental abnormalities within the brains' visual pathway: specifically disorganization, blending of layers, laminar discontinuities, and regions of low cell density within the LGN. These abnormalities were not observed in the control animals. Our findings demonstrate that the ZIKV's damage to the LGN that occurs during fetal development persists into childhood.

Establishment of neural stem cells from fetal monkey brain for neurotoxicity testing.

Neurotoxicity assessments are generally performed using laboratory animals. However, as in vitro neurotoxicity models are continuously refined to reach adequate predicative concordance with in vivo responses, they are increasingly used for some endpoints of neurotoxicity. In this study, gestational day 80 fetal rhesus monkey brain tissue was obtained for neural stem cells (NSCs) isolation. Cells from the entire hippocampus were harvested, mechanically dissociated, and cultured for proliferation and differentiation. Immunocytochemical staining and biological assays demonstrated that the harvested hippocampal cells exhibited typical NSC phenotypes in vitro: (1) cells proliferated vigorously and expressed NSC markers nestin and sex-determining region Y-box 2 (SOX2) and (2) cells differentiated into neurons, astrocytes, and oligodendrocytes, as confirmed by positive staining with class III β-tubulin, glial fibrillary acidic protein, and galactocerebroside, respectively. The NSC produced detectable responses following neurotoxicant exposures (e.g. trimethyltin and 3-nitropropionic acid). Our results indicated that non-human primate NSCs may be a practical tool to study the biology of neural cells and to evaluate the neurotoxicity of chemicals in vitro, thereby providing data that are translatable to humans and may also reduce the number of animals needed for developmental neurotoxicological studies.

CHD8 mutations increase gliogenesis to enlarge brain size in the nonhuman primate

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition that affects social interaction and behavior. Mutations in the gene encoding chromodomain helicase DNA-binding protein 8 (CHD8) lead to autism symptoms and macrocephaly by a haploinsufficiency mechanism. However, studies of small animal models showed inconsistent findings about the mechanisms for CHD8 deficiency-mediated autism symptoms and macrocephaly. Using the nonhuman primate as a model system, we found that CRISPR/Cas9-mediated CHD8 mutations in the embryos of cynomolgus monkeys led to increased gliogenesis to cause macrocephaly in cynomolgus monkeys. Disrupting CHD8 in the fetal monkey brain prior to gliogenesis increased the number of glial cells in newborn monkeys. Moreover, knocking down CHD8 via CRISPR/Cas9 in organotypic monkey brain slices from newborn monkeys also enhanced the proliferation of glial cells. Our findings suggest that gliogenesis is critical for brain size in primates and that abnormal gliogenesis may contribute to ASD.

Robust engraftment of fetal nonhuman primate CD34-positive cells in immune-deficient mice.

Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high-throughput in vivo experimentation leveraged to inform large animal immunology-based studies. We found that adult rhesus macaque mobilized blood (AMb) CD34+-enriched hematopoietic stem and progenitor cells (HSPCs) engrafted at low but persistent levels in immune-deficient mice harboring transgenes for human (NHP cross-reactive) GM-CSF and IL3, but did not in mice with wild-type murine cytokines lacking NHP cross-reactivity. To enhance engraftment, fetal liver-derived HSPCs were selected as the infusion product based on an increased CD34hi fraction compared with AMb and bone marrow. Coupled with cotransplantation of rhesus fetal thymic fragments beneath the mouse kidney capsule, fetal liver-derived HSPC infusion in cytokine-transgenic mice yielded robust multilineage lymphohematopoietic engraftment. The emergent immune system recapitulated that of the fetal monkey, with similar relative frequencies of lymphocyte, granulocyte, and monocyte subsets within the thymic, secondary lymphoid, and peripheral compartments. Importantly, while exhibiting a predominantly naïve phenotype, in vitro functional assays demonstrated robust cellular activation in response to nonspecific and allogenic stimuli. This primatized mouse represents a viable and translatable model for the study of hematopoietic stem cell physiology, immune development, and functional immunology in NHPs. Summary Sentence: Engraftment of rhesus macaque hematopoietic tissues in immune-deficient mice yields a robust BLT/NeoThy-type primatized mouse model for studying nonhuman primate hematopoiesis and immune function in vivo.

PINK1 kinase dysfunction triggers neurodegeneration in the primate brain without impacting mitochondrial homeostasis

In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson’s disease (PD). However, difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1. Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains. CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology. Importantly, PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival. Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD.

Single-cell RNA sequencing reveals regulation of fetal ovary development in the monkey (Macaca fascicularis)

Germ cells are vital for reproduction and heredity. However, the mechanisms underlying female germ cell development in primates, especially in late embryonic stages, remain elusive. Here, we performed single-cell RNA sequencing of 12,471 cells from whole fetal ovaries, and explored the communications between germ cells and niche cells. We depicted the two waves of oogenesis at single-cell resolution and demonstrated that progenitor theca cells exhibit similar characteristics to Leydig cells in fetal monkey ovaries. Notably, we found that ZGLP1 displays differentially expressed patterns between mouse and monkey, which is not overlapped with NANOG in monkey germ cells, suggesting its role in meiosis entry but not in activating oogenic program in primates. Furthermore, the majority of germ cell clusters that sharply express PRDM9 and SPO11 might undergo apoptosis after cyst breakdown, leading to germ cell attrition. Overall, our work provides new insights into the molecular and cellular basis of primate fetal ovary development at single-cell resolution.

Generation of cynomolgus monkey fetuses with intracytoplasmic sperm injection based on the MII-stage oocytes acquired by personalized superovulation protocol.

BackgroundMature oocytes at the metaphase II status (MII-stage oocytes) played an important role in assisted reproductive technology in non-human primates.ObjectivesIn order to improve the proportion of MII-stage oocytes retrieval, three different superovulation protocols were performed on 24 female cynomolgus monkeys.MethodsAll the monkeys received once-daily injection of follicle-stimulating hormone (25 international unit [IU]) on day 3 of the menstruation, 3-day intervals, twice daily for 8–12 days until the time of human chorionic gonadotropin (1,500 IU) injection, on the 14–17th day of menstruation collecting oocytes. The difference between protocol I and protocol II was that 0.1 mg the gonadotropin-releasing hormone agonist was injected on day 1 of the menstruation, while the difference between personalized superovulation protocol and protocol II was that oocytes could be collected on the 14–17th day of menstrual cycle according to the length of each monkey.ResultsThe total number of oocytes harvested using the personalized superovulation protocol was much higher than that using protocol I (p < 0.05), and the proportion of MII-stage oocytes was significantly greater than that from either superovulation protocol I or II (p < 0.001 and p < 0.01 respectively), while the proportion of immature oocytes at the germinal vesicle was less than that from superovulation protocol I (p < 0.05).ConclusionsThe personalized superovulation protocol could increase the rate of MII-stage oocytes acquired, and successfully develop into embryos after intracytoplasmic sperm injection, and eventually generated fetus.

Improving Cell Survival in Injected Embryos Allows Primed Pluripotent Stem Cells to Generate Chimeric Cynomolgus Monkeys

Monkeys are an optimal model species for developing stem cell therapies. We previously reported generating chimeric cynomolgus monkey fetuses using dome-shaped embryonic stem cells (dESCs). However, conventional primed pluripotent stem cells (pPSCs) lack chimera competency. Here, by altering the media in which injected morulae are cultured, weobserved increased survival of cynomolgus monkey primed ESCs, induced PSCs, and somatic cell nuclear transfer-derived ESCs, thereby enabling chimeric contributions with 0.1%-4.5% chimerism into the embryonic and placental tissues, including germ cell progenitors in chimeric monkeys. Mechanically, dESCs and pPSCs belong to different cell types and similarly express epiblast ontogenic genes. The host embryonic microenvironment could reprogram injected PSCs to embryonic-like cells. However, the reprogramming level and chimerism were associated with the cell state of injected PSCs. Our findings provide a method to understand pluripotency and broaden the use of embryonic chimeras for basic developmental biology research and regenerative medicine.

Establishment of an in vitro cytotoxicity assay platform using primary monkey cardiomyocytes

To establish an in vitro cytotoxicity assay platform using monkey cardiomyocytes, we isolated primary cardiomyocytes from fetal cynomolgus monkeys at different gestation days (from day 39 to 90) using the trypsin and collagenase digestion method, which was identical to the standard procedure for rat cardiomyocytes. Under these conditions, the primary cells obtained from monkeys at gestation day 63 or earlier showed spontaneous beating, with >80% cells being viable from all fetuses. Transcriptome analysis of the monkey cardiomyocytes indicated that the cells have essential components of cardiac functions, such as myosins, α-actin, cardiac troponins, and calcium-related molecules. The susceptibility to doxorubicin-induced cytotoxicity in monkey cardiomyocytes was comparable to that in rat cardiomyocytes, as evaluated based on intracellular ATP levels. Microarray analysis with Ingenuity Pathway Analysis revealed that doxorubicin predominantly increased the expression of several key genes involved in the endoplasmic reticulum stress pathway in monkey cardiomyocytes than in rat cardiomyocytes. In conclusion, we isolated primary monkey cardiomyocytes that showed similar sensitivity to doxorubicin as compared with rat cardiomyocytes. This in vitro monkey cardiomyocyte assay platform would serve as a powerful tool for the investigation of the interspecies differences in drug-induced cardiotoxicity and its underlying mechanism.

A cure in the womb

Emboldened by recent success stories of human gene therapy trials in children and infants, researchers are now pushing to treat diseases before birth. Some scientists are excited by the idea of using fetal gene therapy to fix genetic conditions that can’t be treated after birth, but others raise questions about the technique’s feasibility and practicality in humans. A newly published study demonstrates that surgically injecting DNA-stuffed viruses into mice can treat genetic diseases in the womb, when the potential for improvement is the most dramatic. It shows that the surgery is possible in fetal monkeys as well. “For many genetic diseases, the damage starts early,” says Simon N. Waddington, a researcher at University College London who led the new study on an experimental gene therapy for Gaucher disease. The disease is the most common lysosomal storage disorder, a group of metabolic conditions in which an enzyme deficiency results in toxic

Microglia: An Intrinsic Component of the Proliferative Zones in the Fetal Rhesus Monkey (Macaca mulatta) Cerebral Cortex.

Microglial cells are increasingly recognized as modulators of brain development. We previously showed that microglia colonize the cortical proliferative zones in the prenatal brain and regulate the number of precursor cells through phagocytosis. To better define cellular interactions between microglia and proliferative cells, we performed lentiviral vector-mediated intraventricular gene transfer to induce enhanced green fluorescent protein expression in fetal cerebrocortical cells. Tissues were collected and counterstained with cell-specific markers to label microglial cells and identify other cortical cell types. We found that microglial cells intimately interact with the radial glial scaffold and make extensive contacts with neural precursor cells throughout the proliferative zones, particularly in the rhesus monkey fetus when compared to rodents. We also identify a subtype of microglia, which we term 'periventricular microglia', that interact closely with mitotic precursor cells in the ventricular zone. Our data suggest that microglia are structural modulators that facilitate remodeling of the proliferative zones as precursor cells migrate away from the ventricle and may facilitate the delamination of precursor cells. Taken together, these results indicate that microglial cells are an integral component of cortical proliferative zones and contribute to the interactive milieu in which cortical precursor cells function.

Placental and Fetal Effects of Onartuzumab, a Met/HGF Signaling Antagonist, When Administered to Pregnant Cynomolgus Monkeys.

Onartuzumab is an engineered single arm, monovalent monoclonal antibody that targets the MET receptor and prevents hepatocyte growth factor (HGF) signaling. Knockout mice have clearly demonstrated that HGF/MET signaling is developmentally critical. A pre- and postnatal development study (enhanced design) was conducted in cynomolgus monkeys to evaluate the potential developmental consequences following onartuzumab administration. Control or onartuzumab, at loading/maintenance doses of 75/50 mg/kg (low) or 100/100 mg/kg (high), was administered intravenously once weekly to 12 confirmed pregnant female cynomolgus monkeys per group from gestation day (GD) 20 through GD 174. Onartuzumab administration resulted in decreased gestation length, decreased birth weight, and increased fetal and perinatal mortality. A GD147 C-section was conducted for a subset of Control and High Dose monkeys, and identified placental infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate. These findings were limited to placentas from onartuzumab-treated animals. In addition, decreased cellularity of the hepatocytes with dilated hepatic sinusoids was inconsistently observed in the liver of a few fetal or infant monkeys that died in the perinatal period. Surviving offspring had some evidence of developmental delay compared with controls, but no overt teratogenicity. Overall, effects on the perinatal fetuses were consistent with those reported in knockout mice, but not as severe. Onartuzumab concentrations were low or below the level of detection in most offspring, with cord blood concentrations only 1%-2% of maternal levels on GD 147. Malperfusion secondary to onartuzumab-induced placental injury could explain the adverse pregnancy outcomes, fetal growth restriction and relatively low fetal exposures.

Cloning of Macaque Monkeys by Somatic Cell Nuclear Transfer

Generation of genetically uniform non-human primates may help to establish animal models for primate biology and biomedical research. In this study, we have successfully cloned cynomolgus monkeys (Macaca fascicularis) by somatic cell nuclear transfer (SCNT). We found that injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at one-cell stage following SCNT greatly improved blastocyst development and pregnancy rate of transplanted SCNT embryos in surrogate monkeys. For SCNT using fetal monkey fibroblasts, 6 pregnancies were confirmed in 21 surrogates and yielded 2healthy babies. For SCNT using adult monkey cumulus cells, 22 pregnancies were confirmed in 42surrogates and yielded 2 babies that were short-lived. In both cases, genetic analyses confirmed that the nuclear DNA and mitochondria DNA of the monkey offspring originated from the nucleus donor cell and the oocyte donor monkey, respectively. Thus, cloning macaque monkeys by SCNT is feasible using fetal fibroblasts.

Small head size and delayed body weight growth in wild Japanese monkey fetuses after the Fukushima Daiichi nuclear disaster

To evaluate the biological effect of the Fukushima Daiichi nuclear disaster, relative differences in the growth of wild Japanese monkeys (Macaca fuscata) were measured before and after the disaster of 2011 in Fukushima City, which is approximately 70 km from the nuclear power plant, by performing external measurements on fetuses collected from 2008 to 2016. Comparing the relative growth of 31 fetuses conceived prior to the disaster and 31 fetuses conceived after the disaster in terms of body weight and head size (product of the occipital frontal diameter and biparietal diameter) to crown-rump length ratio revealed that body weight growth rate and proportional head size were significantly lower in fetuses conceived after the disaster. No significant difference was observed in nutritional indicators for the fetuses’ mothers. Accordingly, radiation exposure could be one factor contributed to the observed growth delay in this study.

Regional difference in sulcal infolding progression correlated with cerebral cortical expansion in cynomolgus monkey fetuses.

The present study aimed to specify the cerebral sulci developed by cortical expansion in cynomolgus monkey fetuses. The degree of sulcal infolding was evaluated by the gyrification index (GI), which was quantified using ex vivo magnetic resonance imaging. The correlation of cortical volume with the sulcal GI was most frequent during embryonic days (EDs) 100 to 120. Interestingly, the high correlation was marked during EDs 140 to 150 in restricted primary sulci in prefrontal, parietotemporal and medial temporal regions. The present results suggest that cortical expansion is involved in gyral demarcation by sulcal infolding, followed by the sulcal infolding progression in phylogenetically-newer cortices.

Transfer of Therapeutic Genes into Fetal Rhesus Monkeys Using Recombinant Adeno-Associated Type I Viral Vectors.

Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa-/- mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 1012 particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects.

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/β-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.