RESUMEN
Safety and efficacy of therapeutic antibodies are often dependent on their interaction with Fc receptors for IgG (FcγRs). The Göttingen minipig represents a valuable species for biomedical research but its use in preclinical studies with therapeutic antibodies is hampered by the lack of knowledge about the porcine FcγRs. Genome analysis and sequencing now enabled the localization of the previously described FcγRIIIa in the orthologous location to human FCGR3A. In addition, we identified nearby the gene coding for the hitherto undescribed putative porcine FcγRIIa. The 1'241 bp long FCGR2A cDNA translates to a 274aa transmembrane protein containing an extracellular region with high similarity to human and cattle FcγRIIa. Like in cattle, the intracellular part does not contain an immunoreceptor tyrosine-based activation motif (ITAM) as in human FcγRIIa. Flow cytometry of the whole blood and single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) of Göttingen minipigs revealed the expression profile of all porcine FcγRs which is compared to human and mouse. The new FcγRIIa is mainly expressed on platelets making the minipig a good model to study IgG-mediated platelet activation and aggregation. In contrast to humans, minipig blood monocytes were found to express inhibitory FcγRIIb that could lead to the underestimation of FcγR-mediated effects of monocytes observed in minipig studies with therapeutic antibodies.
Asunto(s)
Receptores de IgG/genética , Porcinos Enanos/inmunología , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Ratones , Receptores de IgG/análisis , Receptores de IgG/química , PorcinosRESUMEN
The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established.
Asunto(s)
Blastocisto/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Animales , Blastocisto/citología , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Endodermo/citología , Endodermo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Mórula/citología , Mórula/metabolismo , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Oocitos/citología , Oocitos/metabolismo , Embarazo , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Cigoto/citología , Cigoto/metabolismoRESUMEN
Primordial germ cells (PGCs) and somatic cells originate from postimplantation epiblast cells in mice. As pluripotency is lost upon differentiation of somatic lineages, a naive epigenome and the pluripotency network are re-established during PGC development. Here we demonstrate that Prdm14 contributes not only to PGC specification, but also to naive pluripotency in embryonic stem (ES) cells by repressing the DNA methylation machinery and fibroblast growth factor (FGF) signalling. This indicates a critical role for Prdm14 in programming PGCs and promoting pluripotency in ES cells.
Asunto(s)
Células Madre Embrionarias/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/genética , Animales , Diferenciación Celular , Metilación de ADN , Proteínas de Unión al ADN , Células Madre Embrionarias/citología , Factores de Crecimiento de Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células Germinativas/citología , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células Madre Pluripotentes/citología , Proteínas de Unión al ARN , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Tuberous sclerosis complex (TSC) is a genetic disease characterized by benign tumor growths in multiple organs and neurological symptoms induced by mTOR hyperfunction. Because the molecular pathology is highly complex and the etiology poorly understood, we employed a defined human neuronal model with a single mTOR activating mutation to dissect the disease-relevant molecular responses driving the neuropathology and suggest new targets for treatment. METHODS: We investigate the disease phenotype of TSC by neural differentiation of a human stem cell model that had been deleted for TSC2 by genome editing. Comprehensive genomic analysis was performed by RNA sequencing and ribosome profiling to obtain a detailed genome-wide description of alterations on both the transcriptional and translational level. The molecular effect of mTOR inhibitors used in the clinic was monitored and comparison to published data from patient biopsies and mouse models highlights key pathogenic processes. RESULTS: TSC2-deficient neural stem cells showed severely reduced neuronal maturation and characteristics of astrogliosis instead. Transcriptome analysis indicated an active inflammatory response and increased metabolic activity, whereas at the level of translation ribosomal transcripts showed a 5'UTR motif-mediated increase in ribosome occupancy. Further, we observed enhanced protein synthesis rates of angiogenic growth factors. Treatment with mTOR inhibitors corrected translational alterations but transcriptional dysfunction persisted. CONCLUSIONS: Our results extend the understanding of the molecular pathophysiology of TSC brain lesions, and suggest phenotype-tailored pharmacological treatment strategies.
RESUMEN
Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Inhibition of MEK and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.
Asunto(s)
Metilación de ADN , Células Madre Embrionarias/fisiología , Células Germinativas/citología , Células Madre Pluripotentes/fisiología , Animales , Benzamidas/farmacología , Diferenciación Celular , Células Cultivadas/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Proteínas de Unión al ADN , Difenilamina/análogos & derivados , Difenilamina/farmacología , Inhibidores Enzimáticos/farmacología , Epigénesis Genética , Perfilación de la Expresión Génica , Impresión Genómica , Células Germinativas/fisiología , Estratos Germinativos/citología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Madre Pluripotentes/citología , Piridinas/farmacología , Pirimidinas/farmacología , Proteínas de Unión al ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma , ADN Metiltransferasa 3BRESUMEN
Development of mammalian primordial germ cells (PGCs) presents a unique example of a cell fate specification event that is intimately linked with epigenetic reprogramming. Cell fate commitment is governed by transcription factors which, together with epigenetic regulators, instruct lineage choice in response to signalling cues. Similarly, the reversal of epigenetic silencing is driven by the combinatorial action of transcriptional regulators, resulting in an increase in cellular plasticity. PGCs constitute a paradox, since their development as a unipotent specialised lineage is coupled with extensive reprogramming, which eventually leads to an increase in cellular potency. In this review we discuss the role of key factors in the specification of the germ cell lineage that are also important for the comprehensive erasure of epigenetic modifications, which provides the foundation for regeneration of totipotency. We further discuss current concepts of transcriptional and epigenetic control of cell fate decisions, with a particular focus on emerging principles of enhancer activity and their potential implications for the transcriptional control of PGC specification.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Células Madre Embrionarias/citología , Células Germinativas/metabolismo , Animales , Linaje de la Célula , Metilación de ADN , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Epigenómica , Silenciador del Gen , Humanos , Mamíferos , Transducción de Señal , Factores de TranscripciónRESUMEN
Epigenetic reprogramming in early germ cells is critical toward the establishment of totipotency, but investigations of the germline events are intractable. An objective cell culture-based system could provide mechanistic insight on how the key determinants of primordial germ cells (PGCs), including Prdm14, induce reprogramming in germ cells to an epigenetic ground state. Here we show a Prdm14-Klf2 synergistic effect that can accelerate and enhance reversion of mouse epiblast stem cells (epiSCs) to a naive pluripotent state, including X reactivation and DNA demethylation. Notably, Prdm14 alone has little effect on epiSC reversion, but it enhances the competence for reprogramming and potentially PGC specification. Reprogramming of epiSCs by the combinatorial effect of Prdm14-Klf2 involves key epigenetic changes, which might have an analogous role in PGCs. Our study provides a paradigm toward a systematic analysis of how other key genes contribute to complex and dynamic events of reprogramming in the germline.