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Trophoblast stem cells (TSCs), derived from the trophectoderm of the blastocyst, are used as an in vitro model to reveal the mechanisms underlying placentation in mammals. In humans, suitable culture conditions for TSC derivation have recently been established. The established human TSCs (hTSCs) differentiate efficiently toward two trophoblast subtypes: syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). However, the efficiency of differentiation is lower in macaque TSCs than in hTSCs. Here, we demonstrate that the activation of Wnt signaling downregulated the expression of inhibitory G protein and induced trophoblastic lineage switching to the STB progenitor state. The treatment of macaque TSCs with a GSK-3 inhibitor, CHIR99021, upregulated STB progenitor markers and enhanced proliferation. Under the Wnt signaling-activated conditions, macaque TSCs effectively differentiated to STBs upon dbcAMP and forskolin treatment. RNA-seq analyses revealed the downregulation of inhibitory G protein, which may make macaque TSCs responsive to forskolin. Interestingly, this lineage switching appeared to be reversible as the macaque TSCs lost responsiveness to forskolin upon the removal of CHIR99021. The ability to regulate the direction of macaque TSC differentiation would be advantageous in elucidating the mechanisms underlying placentation in non-human primates.
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A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity. In this study, we examined the potential of phenotypically mixed heterologous dual-pseudotyped LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoproteins, termed V/HN-LV. Our findings demonstrated the significantly improved transduction efficiency of V/HN-LV in various cell lines of mice, cynomolgus monkeys, and humans compared with LV pseudotyped with VSV-G alone. Notably, V/HN-LV showed higher transduction efficiency in human cells, including hematopoietic stem cells. The efficient incorporation of wild-type SeV-HN into V/HN-LV depended on VSV-G. SeV-HN removed sialic acid from VSV-G, and the desialylation of VSV-G increased V/HN-LV infectivity. Furthermore, V/HN-LV acquired the ability to recognize sialic acid, particularly N-acetylneuraminic acid on the host cell, enhancing LV infectivity. Overall, VSV-G and SeV-HN synergistically improve LV transduction efficiency and broaden its tropism, indicating their potential use in gene delivery.
Asunto(s)
Vectores Genéticos , Proteína HN , Lentivirus , Virus Sendai , Transducción Genética , Proteínas del Envoltorio Viral , Animales , Humanos , Vectores Genéticos/genética , Lentivirus/genética , Virus Sendai/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Ratones , Proteína HN/genética , Proteína HN/metabolismo , Línea Celular , Macaca fascicularis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Tropismo Viral , Células HEK293 , Técnicas de Transferencia de Gen , Terapia Genética/métodosRESUMEN
For the advancement of DKD treatment, identifying unrecognized residual risk factors is essential. We explored the impact of obesity diversity derived from different carbohydrate qualities, with an emphasis on the increasing trend of excessive fructose consumption and its effect on DKD progression. In this study, we utilized db/db mice to establish a novel diabetic model characterized by fructose overconsumption, aiming to uncover the underlying mechanisms of renal damage. Compared to the control diet group, the fructose-fed db/db mice exhibited more pronounced obesity yet demonstrated milder glucose intolerance. Plasma cystatin C levels were elevated in the fructose model compared to the control, and this elevation was accompanied by enhanced glomerular sclerosis, even though albuminuria levels and tubular lesions were comparable. Single-cell RNA sequencing of the whole kidney highlighted an increase in Lrg1 in glomerular endothelial cells (GECs) in the fructose model, which appeared to drive mesangial fibrosis through enhanced TGF-ß1 signaling. Our findings suggest that excessive fructose intake exacerbates diabetic kidney disease progression, mediated by aberrant Lrg1-driven crosstalk between GECs and mesangial cells.
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Nefropatías Diabéticas , Células Mesangiales , Ratones , Animales , Células Endoteliales/patología , Fructosa/efectos adversos , Nefropatías Diabéticas/patología , Ratones Endogámicos , Obesidad/complicaciones , Comunicación CelularRESUMEN
Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.
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Mutación con Pérdida de Función , Proteínas de Transporte Vesicular , Animales , Ratones , Humanos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Células Endoteliales/metabolismo , Secreción de Insulina , Colesterol , Mamíferos/metabolismoRESUMEN
Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder that is associated with a high plasma level of low-density lipoprotein (LDL) cholesterol, leading to an increased risk of cardiovascular diseases. To develop basic and translational research on FH, we here generated an FH model in a non-human primate (cynomolgus monkeys) by deleting the LDL receptor (LDLR) gene using the genome editing technique. Six LDLR knockout (KO) monkeys were produced, all of which were confirmed to have mutations in the LDLR gene by sequence analysis. The levels of plasma cholesterol and triglyceride were quite high in the monkeys, and were similar to those in FH patients with homozygous mutations in the LDLR gene. In addition, periocular xanthoma was observed only 1 year after birth. Lipoprotein profile analysis showed that the plasma very low-density lipoprotein and LDL were elevated, while the plasma high density lipoprotein was decreased in LDLR KO monkeys. The LDLR KO monkeys were also strongly resistant to medications for hypercholesterolemia. Taken together, we successfully generated a non-human primate model of hypercholesterolemia in which the phenotype is similar to that of homozygous FH patients.
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Traumatismos Craneocerebrales , Hipercolesterolemia , Hiperlipoproteinemia Tipo II , Animales , Humanos , Primates , Hiperlipoproteinemia Tipo II/genética , Lipoproteínas LDL , Macaca fascicularisRESUMEN
Background: The placenta is an extraembryonic organ, which is essential to maintain a normal pregnancy. However, placental development in humans is poorly understood because of technical and ethical reasons. Methods: We analyzed the anatomical localization of each trophoblastic subtype in the cynomolgus monkey placenta by immunohistochemistry in the early second trimester. Histological differences among the mouse, cynomolgus monkey, and human placenta were compared. The PubMed database was used to search for studies on placentation in rodents and primates. Main findings: The anatomical structures and subtypes of the placenta in cynomolgus monkeys are highly similar to those in humans, with the exception of fewer interstitial extravillous trophoblasts in cynomolgus monkeys. Conclusion: The cynomolgus monkey appears to be a good animal model to investigate human placentation.
RESUMEN
Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.
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Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine-placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal-fetal interface.
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Lipoproteínas/metabolismo , Placentación/fisiología , Células Madre/fisiología , Trofoblastos/fisiología , Animales , Sistemas CRISPR-Cas , Células Endoteliales/fisiología , Femenino , Edición Génica , Humanos , Lipoproteínas/genética , Mutación , Placenta/metabolismo , Embarazo , Interferencia de ARN , Ratas , Ratas Sprague-DawleyRESUMEN
The placenta forms a maternal-fetal junction that supports many physiological functions such as the supply of nutrition and exchange of gases and wastes. Establishing an in vitro culture model of human and non-human primate trophoblast stem/progenitor cells is important for investigating the process of early placental development and trophoblast differentiation. In this study, we have established five trophoblast stem cell (TSC) lines from cynomolgus monkey blastocysts, named macTSC #1-5. Fibroblast growth factor 4 (FGF4) enhanced proliferation of macTSCs, while other exogenous factors were not required to maintain their undifferentiated state. macTSCs showed a trophoblastic gene expression profile and trophoblast-like DNA methylation status and also exhibited differentiation capacity towards invasive trophoblast cells and multinucleated syncytia. In a xenogeneic chimera assay, these stem cells contributed to trophectoderm (TE) development in the chimeric blastocysts. macTSC are the first primate trophoblast cell lines whose proliferation is promoted by FGF4. These cell lines provide a valuable in vitro culture model to analyze the similarities and differences in placental development between human and non-human primates.
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Técnicas de Cultivo de Célula/métodos , Células Madre/citología , Trofoblastos/citología , Animales , Bucladesina/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Quimera , Cromosomas de los Mamíferos/genética , Metilación de ADN/genética , Ectodermo/citología , Regulación de la Expresión Génica/efectos de los fármacos , Células Gigantes/citología , Macaca fascicularis , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Especificidad de la Especie , Células Madre/efectos de los fármacos , Trofoblastos/efectos de los fármacosRESUMEN
DNA methylation is an epigenetic modification important for cell fate determination and cell type-specific gene expression. Transcriptional regulatory regions of the mammalian genome contain a large number of tissue/cell type-dependent differentially methylated regions (T-DMRs) with DNA methylation patterns crucial for transcription of the corresponding genes. In general, tissues consist of multiple cell types in various proportions, making it difficult to detect T-DMRs of minor cell types in tissues. The present study attempts to detect T-DMRs of minor cell types in tissues by ultra-deep bisulfite sequencing of cell type-restricted genes and to assume proportions of minor cell types based on DNA methylation patterns of sequenced reads. For this purpose, we focused on transcriptionally active hypomethylated alleles (Hypo-alleles), which can be recognized by the high ratio of unmethylated CpGs in each sequenced read (allele). The pituitary gland contains multiple cell types including five hormone-expressing cell types and stem/progenitor cells, each of which is a minor cell type in the pituitary tissue. By ultra-deep sequencing of more than 100 reads for detection of Hypo-alleles in pituitary cell type-specific genes, we identified T-DMRs specific to hormone-expressing cells and stem/progenitor cells and used them to estimate the proportions of each cell type based on the Hypo-allele ratio in pituitary tissue. Therefore, introduction of the novel Hypo-allele concept enabled us to detect T-DMRs of minor cell types with estimation of their proportions in the tissue by ultra-deep bisulfite sequencing.