Decoding host-microbiome interactions through co-expression network analysis within the non-human primate intestine.

The gut microbiome affects the health status of the host through complex interactions with the host's intestinal wall. These host-microbiome interactions may spatially vary along the physical and chemical environment of the intestine, but these changes remain unknown. This study investigated these intricate relationships through a gene co-expression network analysis based on dual transcriptome profiling of different intestinal sites-cecum, transverse colon, and rectum-of the primate common marmoset. We proposed a gene module extraction algorithm based on the graph theory to find tightly interacting gene modules of the host and the microbiome from a vast co-expression network. The 27 gene modules identified by this method, which include both host and microbiome genes, not only produced results consistent with previous studies regarding the host-microbiome relationships, but also provided new insights into microbiome genes acting as potential mediators in host-microbiome interplays. Specifically, we discovered associations between the host gene FBP1, a cancer marker, and polysaccharide degradation-related genes (pfkA and fucI) coded by Bacteroides vulgatus, as well as relationships between host B cell-specific genes (CD19, CD22, CD79B, and PTPN6) and a tryptophan synthesis gene (trpB) coded by Parabacteroides distasonis. Furthermore, our proposed module extraction algorithm surpassed existing approaches by successfully defining more functionally related gene modules, providing insights for understanding the complex relationship between the host and the microbiome.IMPORTANCEWe unveiled the intricate dynamics of the host-microbiome interactions along the colon by identifying closely interacting gene modules from a vast gene co-expression network, constructed based on simultaneous profiling of both host and microbiome transcriptomes. Our proposed gene module extraction algorithm, designed to interpret inter-species interactions, enabled the identification of functionally related gene modules encompassing both host and microbiome genes, which was challenging with conventional modularity maximization algorithms. Through these identified gene modules, we discerned previously unrecognized bacterial genes that potentially mediate in known relationships between host genes and specific bacterial species. Our findings underscore the spatial variations in host-microbiome interactions along the colon, rather than displaying a uniform pattern throughout the colon.

Hypoblast from human pluripotent stem cells regulates epiblast development.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

mRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells.

Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by in vitro culture or in vivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLCs. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development.

A Case of Primary Intraosseous Adenoid Cystic Carcinoma of the Mandible.

Primary intraosseous adenoid cystic carcinoma (PIACC) of the jaw is rare. To our knowledge, only 51 cases have been reported in the English literature. We present a rare case of PIACC arising in the mandible with multiple bone metastases and review the previous articles. A 70-year-old woman presented with paresthesia of the right chin and lower gingiva for 4 months. Radiography revealed an irregular radiolucent region on the right side of the ramus, infiltrating to the mandibular canal. Biopsy revealed a pathological diagnosis of adenoid cystic carcinoma. Multiple bone metastases were present in the sternum, scapula, and thighs. The treatment effect was progressive disease for chemotherapy; therefore, best supportive care was provided for 3 years.

The common marmoset in biomedical research: experimental disease models and veterinary management.

The common marmoset, Callithrix jacchus, is increasingly being used as the preferred nonhuman primate (NHP) model in biomedical research. Marmosets share several physiological and biological similarities with humans, as a Simiiformes species, and their use in research programs advances knowledge in several fields. Their unique characteristics, such as their small size, high fecundity, and rapid growth, offer additional advances in laboratory settings. This article reviews the developments in experimental disease models using marmosets based on our experience at the Central Institute for Experimental Animals (CIEA) in Japan. The development of genetically modified marmoset models using advanced genome editing technology is attracting researchers, particularly in neuroscience-related fields. In parallel, various marmoset models of human diseases induced by surgery or drug administration have contributed to preclinical and translational studies. Among these are models for Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, spinal cord injury models, a model for type 1 diabetes induced by the combination of partial pancreatectomy and streptozotocin administration, and a hepatic fibrosis model induced by thioacetamide. The development of these models has been supported by refinements in veterinary care, such as the careful design of anesthetic protocols and better understanding of pathogenic microorganisms. In the second part of this review, we present a compilation of practices currently in use at CIEA that provide optimal animal care and enable safe experimentation.

Effects of Wnt-ß-Catenin Signaling and Sclerostin on the Phenotypes of Rat Pheochromocytoma PC12 Cells.

Pheochromocytomas and paragangliomas (PPGLs) are classified into 3 major categories with distinct driver genes: pseudohypoxia, kinase signaling, and Wnt-altered subtypes. PPGLs in the Wnt-altered subtype are sporadic and tend to be aggressive with metastasis, where somatic gene fusions affecting mastermind-like 3 (MAML3) and somatic mutations in cold shock domain containing E1 (CSDE1) cause overactivation of Wnt-ß-catenin signaling. However, the relation between Wnt-ß-catenin signaling and the biological behavior of PPGLs remains unexplored. In rat pheochromocytoma PC12 cells, Wnt3a treatment enhanced cell proliferation and suppressed mRNA expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis, and dopamine secretion. We identified the expression of sclerostin in PC12 cells, which is known as an osteocyte-derived negative regulator for Wnt signaling-driven bone formation. Inhibition of endogenous Wnt pathway by XAV939 or sclerostin resulted in attenuated cell proliferation and increased TH expression. Furthermore, Wnt3a pretreatment suppressed bone morphogenetic protein (BMP)-induced Smad1/5/9 phosphorylation whereas BMPs enhanced sclerostin expression in PC12 cells. In the Wnt-altered subtype, the increased Wnt-ß-catenin pathway may contribute the aggressive clinical behavior with reduced catecholamine production. Furthermore, upregulated expression of sclerostin by BMPs may explain the osteolytic metastatic lesions observed in metastatic PPGLs.

Immortalization of common marmoset-derived fibroblasts via expression of cell cycle regulators using the piggyBac transposon.

Common marmosets are non-human primate models used in biomedical research and genome editing technology. This study aimed to establish cell lines from common marmosets and evaluate their characteristics. We obtained normal fibroblasts derived from muscle tissues of two common marmosets and immortalized them with the introduction of a mutat form of cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomere reverse transcriptase (TERT) using the piggyBac transposon. Compared to parental cells, the immortalized cell lines (named K4DT cells) showed telomerase activity and an accelerated cell proliferation rate. To our knowledge, this is the first study describing the successful establishment of immortalized common marmoset-derived fibroblasts using piggyBac transposition of CDK4R24C, Cyclin D1, and TERT. Our generated cell lines might be a beneficial tool for future studies on disease modeling and targeted gene therapies.

Recent Advances in the Modeling of Alzheimer's Disease.

Since 1995, more than 100 transgenic (Tg) mouse models of Alzheimer's disease (AD) have been generated in which mutant amyloid precursor protein (APP) or APP/presenilin 1 (PS1) cDNA is overexpressed ( 1st generation models ). Although many of these models successfully recapitulate major pathological hallmarks of the disease such as amyloid ß peptide (Aß) deposition and neuroinflammation, they have suffered from artificial phenotypes in the form of overproduced or mislocalized APP/PS1 and their functional fragments, as well as calpastatin deficiency-induced early lethality, calpain activation, neuronal cell death without tau pathology, endoplasmic reticulum stresses, and inflammasome involvement. Such artifacts bring two important uncertainties into play, these being (1) why the artifacts arise, and (2) how they affect the interpretation of experimental results. In addition, destruction of endogenous gene loci in some Tg lines by transgenes has been reported. To overcome these concerns, single App knock-in mouse models harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice) were developed ( 2nd generation models ). While these models are interesting given that they exhibit Aß pathology, neuroinflammation, and cognitive impairment in an age-dependent manner, the model with the Artic mutation, which exhibits an extensive pathology as early as 6 months of age, is not suitable for investigating Aß metabolism and clearance because the Aß in this model is resistant to proteolytic degradation and is therefore prone to aggregation. Moreover, it cannot be used for preclinical immunotherapy studies owing to the discrete affinity it shows for anti-Aß antibodies. The weakness of the latter model (without the Arctic mutation) is that the pathology may require up to 18 months before it becomes sufficiently apparent for experimental investigation. Nevertheless, this model was successfully applied to modulating Aß pathology by genome editing, to revealing the differential roles of neprilysin and insulin-degrading enzyme in Aß metabolism, and to identifying somatostatin receptor subtypes involved in Aß degradation by neprilysin. In addition to discussing these issues, we also provide here a technical guide for the application of App knock-in mice to AD research. Subsequently, a new double knock-in line carrying the AppNL-F and Psen1 P117L/WT mutations was generated, the pathogenic effect of which was found to be synergistic. A characteristic of this 3rd generation model is that it exhibits more cored plaque pathology and neuroinflammation than the AppNL-G-F line, and thus is more suitable for preclinical studies of disease-modifying medications targeting Aß. Furthermore, a derivative AppG-F line devoid of Swedish mutations which can be utilized for preclinical studies of ß-secretase modifier(s) was recently created. In addition, we introduce a new model of cerebral amyloid angiopathy that may be useful for analyzing amyloid-related imaging abnormalities that can be caused by anti-Aß immunotherapy. Use of the App knock-in mice also led to identification of the α-endosulfine-K ATP channel pathway as components of the somatostatin-evoked physiological mechanisms that reduce Aß deposition via the activation of neprilysin. Such advances have provided new insights for the prevention and treatment of preclinical AD. Because tau pathology plays an essential role in AD pathogenesis, knock-in mice with human tau wherein the entire murine Mapt gene has been humanized were generated. Using these mice, the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) was discovered as a mediator linking tau pathology to neurodegeneration and showed that tau humanization promoted pathological tau propagation. Finally, we describe and discuss the current status of mutant human tau knock-in mice and a non-human primate model of AD that we have successfully created.

Spontaneous pulmonary adenocarcinoma in a common marmoset (Callithrix jacchus).

A seven-year-old female common marmoset (Callithrix jacchus) presented with weight loss. Imaging revealed a left thoracic mass, confirmed at necropsy. Histology and immunohistochemistry suggested a well-differentiated pulmonary adenocarcinoma. No evidence of local lymphovascular invasion or distant metastasis was observed. This is the first report of pulmonary adenocarcinoma in marmosets.

Induction of macrophage-like immunosuppressive cells from common marmoset ES cells by stepwise differentiation with DZNep.

Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) as the resource of therapeutic cells/tissue. However, immune suppression is still needed when the donor-recipient combination is allogeneic. We have reported previously that mouse PSCs-derived immunosuppressive cells contribute to prolonged survival of grafts derived from the same mouse PSCs in allogeneic recipients. For its clinical application, a preclinical study using non-human primates such as common marmoset must be performed. In this study, we established the induction protocol of immunosuppressive cells from common marmoset ES cells. Although similar immunosuppressive macrophages could not be induced by same protocol as that for mouse PSCs, we employed an inhibitor for histone methyltransferase, DZNep, and succeeded to induce them. The DZNep-treated macrophage-like cells expressed several immunosuppressive molecules and significantly inhibited allogeneic mixed lymphocyte reaction. The immunosuppressive cells from non-human primate ESCs will help to establish an immunoregulating strategy in regenerative medicine using PSCs.

Serum anti-recoverin antibodies is found in elderly patients with retinitis pigmentosa and cancer.

PURPOSE: To screen for anti-recoverin antibodies in elderly patients with retinitis pigmentosa (RP) with or without cancer and cross-sectionally characterize the seropositive patients clinically. METHODS: Serum from 75 RP patients who had been tested for mutations in a panel of 83 RP genes and 73 normal controls, all aged 50-80 years, were screened for anti-recoverin antibodies by Western blot using recombinant recoverin, retinal lysate from a marmoset and commercial anti-recoverin antibodies as a control. RESULTS: Three RP patients with typical pigmentary degeneration of the 75 (4.0%) were seropositive for anti-recoverin antibody. Pathogenic mutations were identified in two seropositive RP patients. All three patients had visual impairment since childhood and were diagnosed as RP by the age of 30. The severity of the retinopathy varied greatly among these three patients, ranging in visual acuity from light perception OU to 20/30 OU. Retinitis pigmentosa (RP) patients with a history of cancer were more likely to have anti-recoverin antibodies (3/14; 21.4%) than those without (0/61; 0%; p = 0.005, Fischer exact test). All 73 healthy controls with no history of cancer were also seronegative. CONCLUSION: Our results show that serum anti-recoverin antibodies can be detected in typical RP patients with identified pathogenic mutations and that a history of cancer may increase the risk of developing anti-recoverin antibodies.

A pilot study to establish human T-cell leukemia virus type 1 (HTLV-1) carrier model using common marmoset (Callithrix jacchus).

BACKGROUND: For the diagnosis and treatment of adult T-cell leukemia/lymphoma (ATLL) caused by human T-lymphotropic virus type 1 (HTLV-1) are required therapeutic modalities urgently. Non-human primate models for ATLL would provide a valuable information for clinical studies. We did a pilot study to establish an ATLL non-human primate model using common marmosets (Callithrix jacchus). METHODS: We inoculated HTLV-1-producing MT-2 cells into 9-month-old marmosets, either intraperitoneally or intravenously. We next administrated MT-2 cells into 13-month-old marmosets under cyclosporine A (CsA) treatment to promote infection. HTLV-1 infection was determined by measuring HTLV-1 antibody titer in the common marmosets. RESULTS: The HTLV-1 antibody titer increased in the intraperitoneally inoculated marmoset with or without CsA treatment, and it kept over five 5 years though proviral copy number (proviral load, PVL) remained low throughout the study. CONCLUSION: We obtained HTLV-1 asymptomatic carriers of common marmosets by inoculating MT-2 cells.

Localization of SOX2-positive stem/progenitor cells in the anterior lobe of the common marmoset (Callithrix jacchus) pituitary.

Studies on mouse and rat pituitaries reported that Sox2-expressing cells play roles as stem/progenitor cells in the adult pituitary gland. The presence of cells with stem cell-like properties in the pituitary adenoma and SOX2-positive cells has been demonstrated in the human pituitary. However, considering the difficulty in fully examining the stem/progenitor cell properties in the human pituitary, in the present study, we analyzed the SOX2-positive cells in the pituitary of the adult common marmoset (Callithrix jacchus), which is used as a non-human primate model. Immunohistochemistry demonstrated that localization pattern of SOX2-positive cells in the common marmoset pituitary was similar to that observed in the rodent pituitary, i.e., in the two types of niches (marginal cell layer and parenchymal-niche) and as scattered single cells in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells express S100 and were located in the center or interior of LAMININ-positive micro-lobular structures. Collectively, the present study reveals properties of SOX2-positive cells in the common marmoset pituitary and suggests that the common marmoset proves to be a useful tool for analyzing pituitary stem/progenitor cells in a non-human primate model.

Levels of Anti-Retinal Antibodies in Retinal Detachment and Proliferative Vitreoretinopathy.

PURPOSE: The purpose of the study is to investigate the correlation between intraocular anti-retinal antibodies and clinical measurements in patients with rhegmatogenous retinal detachment (RRD) and proliferative vitreoretinopathy (PVR). MATERIAL AND METHODS: Aqueous humor and vitreous samples were collected from patients with RRD, PVR, and from control subjects with macular hole. The levels of total protein (TP), IgG, and anti-retinal antibodies were determined with a bicinchoninic acid assay, enzyme-linked immunosorbent assay, and dot blot, respectively. Correlations between these measurements were assessed using Pearson's correlation test. Analysis of variance followed by a post-hoc test or the Student t-test was used to compare differences between groups. RESULTS: The levels of anti-retinal antibodies and IgG were correlated with each other (P < 0.010). The IgG concentration was higher in patients with PVR than in controls in both the aqueous humor (P < 0.001) and the vitreous (P < 0.001), but not in patients with RRD. Conversely, TP levels and anti-retinal antibodies in both ocular fluids from RRD and PVR patients did not significantly differ from the controls. In a subgroup analysis, vitreal anti-retinal antibody levels were correlated with average macular thickness in the re-attached macula following surgery for macula-off RRD/PVR (P = 0.012). Furthermore, patients with post-operative cystoid macular edema had a higher level of vitreal anti-retinal antibodies than those without (P = 0.009). CONCLUSIONS: Intravitreal anti-retinal antibodies were increased in the eyes with maculopathy after surgical intervention for RRD/PVR.

Thioacetamide-induced hepatic fibrosis in the common marmoset.

The common marmoset (Callithrix jacchus) is a nonhuman primate that is used for preclinical research on stem cell transplantation therapies due to its similarity to human beings as well as its small size, enabling researchers to perform experiments without preparing a large number of cells. In this study, we developed a marmoset hepatic fibrosis model for regenerative medicine research. Six female marmosets aged 4-6 years were administered thioacetamide (TAA) at a dose of 2.5-40 mg/kg two or three times a week. Hepatic fibrosis was assessed by liver biopsy when blood chemistry indicated liver damage. Administration of TAA increased total bile acid, aspartate aminotransferase, and total bilirubin and decreased serum albumin levels. Following more than 11 weeks of continuous injection of TAA, histological analyses detected hepatic fibrosis in all animals. Type IV collagen 7S serum levels in animals with hepatic fibrosis were significantly higher than in normal animals as a possible marker of hepatic fibrosis in marmosets. Serial liver biopsies following the last administration of TAA revealed that induced fibrosis remained up to 11 weeks. The results suggest that continuous TAA administration induces persistent hepatic fibrosis in the common marmoset and this nonhuman primate hepatic fibrosis model have the possibility to evaluate the therapeutic effects of test samples to ameliorate hepatic fibrosis.

Comparative immunity of antigen recognition, differentiation, and other functional molecules: similarities and differences among common marmosets, humans, and mice.

The common marmoset (CM; Callithrix jacchus) is a small New World monkey with a high rate of pregnancy and is maintained in closed colonies as an experimental animal species. Although CMs are used for immunological research, such as studies of autoimmune disease and infectious disease, their immunological characteristics are less defined than those of other nonhuman primates. We and others have analyzed antigen recognition-related molecules, the development of hematopoietic stem cells (HSCs), and the molecules involved in the immune response. CMs systemically express Caja-G, a major histocompatibility complex class I molecule, and the ortholog of HLA-G, a suppressive nonclassical HLA class I molecule. HSCs express CD117, while CD34 is not essential for multipotency. CD117+ cells developed into all hematopoietic cell lineages, but compared with human HSCs, B cells did not extensively develop when HSCs were transplanted into an immunodeficient mouse. Although autoimmune models have been successfully established, sensitization of CMs with some bacteria induced a low protective immunity. In CMs, B cells were observed in the periphery, but IgG levels were very low compared with those in humans and mice. This evidence suggests that CM immunity is partially suppressed systemically. Such immune regulation might benefit pregnancy in CMs, which normally deliver dizygotic twins, the placentae of which are fused and the immune cells of which are mixed. In this review, we describe the CM immune system and discuss the possibility of using CMs as a model of human immunity.

Progesterone hydroxylation by cytochromes P450 2C and 3A enzymes in marmoset liver microsomes.

1. Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical drug metabolism studies. However, the roles of marmoset cytochrome P450 (P450) isoforms in the oxidation of endobiotic progesterone have not been fully investigated. In this study, the roles of marmoset P450 isoforms in progesterone hydroxylation were extensively determined. 2. The activities of liver microsomes from individual marmosets with respect to progesterone 21/17α- and 16α/6ß-hydroxylation were significantly correlated with those for flurbiprofen 4-hydroxylation and midazolam 1'-hydroxylation, respectively, as similar correlations have been found in humans. Anti-P450 2 C and 3 A antibodies suppressed progesterone 21/17α- and 16α/6ß-hydroxylation, respectively, in marmoset liver microsomes. 3. Recombinant marmoset P450 2C58 and 2C19 catalyzed progesterone to form 21-hydroxyprogesterone and 16α-hydroxyprogesterone, respectively, as major products with high maximum velocity/Km values of 0.53 and 0.089 mL/min/nmol, respectively. Recombinant marmoset P450 3A4/90 oxidized progesterone to form 6ß-hydroxyprogesterone as a major product with homotropic cooperativity (>1 of Hill coefficients). 4. These results indicate that the overall activities and roles of liver microsomal P450 enzymes in marmoset livers are similar to those in humans, especially for progesterone 21/17α- and 16α/6ß-hydroxylation by marmoset P450 2 C and 3 A enzymes, respectively, suggesting important roles for these P450 enzymes in the metabolism of endobiotics in marmosets.

The transcriptomes of novel marmoset monkey embryonic stem cell lines reflect distinct genomic features.

Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP.