RESUMO
The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.
Assuntos
Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/virologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígenos CD55/genética , Antígenos CD55/metabolismo , Linhagem Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Infecções por Coxsackievirus/genética , Ectoderma/metabolismo , Endoderma/metabolismo , Enterovirus Humano B/metabolismo , Enterovirus Humano B/patogenicidade , Perfilação da Expressão Gênica , Camadas Germinativas/citologia , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/virologia , Mesoderma/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , RNA/metabolismoRESUMO
Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I-stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Interferon Tipo I/metabolismo , Antivirais/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/fisiologia , Ectoderma/efeitos dos fármacos , Ectoderma/metabolismo , Ectoderma/fisiologia , Ectoderma/virologia , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Endoderma/fisiologia , Endoderma/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibroblastos/virologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Camadas Germinativas/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/virologia , Fator 4 Semelhante a Kruppel , RNA Viral/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The relative efficiency of retroviral-mediated gene transfer into early passage cultures of different tissues of fetal lamb was investigated. Cells which originate from different embryonic germ layers showed marked differences in infectibility in the descending order: brain (neuroectoderm) > kidney (nephrogenic mesoderm) > muscle (somite mesoderm), lung (endoderm) > skin (ectoderm). In an attempt to generate immortalized brain cell lines, primary cultures were transduced with various oncogenes. A wide variety of morphologically distinct, dividing cells, including neurone-like and glial-like cells, were obtained using some oncogenes but also using the vector without an inserted oncogene.
