Project description:HCMV -treated and control human adult neural precurso cells (NPC) were used to extract RNA for profiling on DNA arrays Primary adult hippocampus-derived neural precursor cells were used at passage # 2-4 for HCMV infection, followed by RNA extraction at indicated times Primary adult neural precursor cells were infected with HCMV strains Towne and TR (O.1MOI) and RNA was extracted at 72 hrs postinfection for expression profiling on both HCMV and Affymetrix DNA arrays
Project description:HCMV treated and control human primary adult neural precursor cells (isolated from hippocampus) were used at passages 2-4 for infection with HCMV and RNA was harvested at indicated times
Project description:HCMV -treated and control human adult neural precurso cells (NPC) were used to extract RNA for profiling on DNA arrays Primary adult hippocampus-derived neural precursor cells were used at passage # 2-4 for HCMV infection, followed by RNA extraction at indicated times
Project description:Congenital human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of mental retardation and congenital deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV has been limited by difficulties in sustaining primary cultures. Neuronal cells derived from human induced pluripotent stem (iPS) cells now provide a novel opportunity to investigate HCMV pathogenesis. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their permissiveness to infection with HCMV strain Ad169. Analysis of iPS cells and nearly pure populations of iPS-derived neural stem cells (NSCs), neuroprogenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; the supernatant from infected neural stem cells and NPCs (but not mock infected cells) induced cytopathic effects in human fibroblasts; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate. Our approach offers powerful cellular models to investigate the effect of neurotropic viral agents on human neurodevelopment. Adherent monolayer culture of neural progenitor cells (NPCs) were either infected with HCMV Ad169 in triplicate, with each individual sample harvested separately to provide biological replicates for expression analysis. Infected and mock-infected cells were harvested 24 h p.i. RNA. NPCs were 70-80% confluence.
Project description:Congenital human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of mental retardation and congenital deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV has been limited by difficulties in sustaining primary cultures. Neuronal cells derived from human induced pluripotent stem (iPS) cells now provide a novel opportunity to investigate HCMV pathogenesis. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their permissiveness to infection with HCMV strain Ad169. Analysis of iPS cells and nearly pure populations of iPS-derived neural stem cells (NSCs), neuroprogenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; the supernatant from infected neural stem cells and NPCs (but not mock infected cells) induced cytopathic effects in human fibroblasts; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate. Our approach offers powerful cellular models to investigate the effect of neurotropic viral agents on human neurodevelopment.
Project description:Congenital human cytomegalovirus (HCMV) infection is the leading infectious cause of birth defects, including neurodevelopmental disorders. HCMV infection mainly targets neural progenitor cells (NPCs) in fetal brains, inducing abnormal differentiation by altering key regulatory pathways. HCMV expresses a series of viral miRNAs during infection, but their roles, particularly in NPCs, are not fully understood. In this study, we characterized expression profiles of both cellular and viral miRNAs in HCMV-infected NPCs by microarray analysis during early infection time points and investigated the primary effects of these miRNAs on regulating NPC fate. While expression of most cellular miRNAs was unaffected by HCMV infection, one cellular miRNA was upregulated and six were downregulated from 2 to 24 h post infection. Moreover, of 17 HCMV miRNAs evaluated, six were differentially expressed in HCMV-infected NPCs during early infection time points.
Project description:We show that HCMV infection of human induced pluripotent stem cell (hiPSC)-derived brain organoids can recapitulate severe clinical manifestations such as microcephaly. We demonstrate that infection of hiPSC-derived brain organoids by the “clinical-like” HCMV strain TB40/E results in substantially reduced brain organoid growth, impaired formation of cortical layers, abnormal calcium signaling, and disrupted neural network. Accordingly, RNA-seq analysis revealed that genes down-regulated in TB40/E-infected brain organoids include those involved in neural development and calcium signaling. Moreover, we show that the impeded brain organoid development and abnormal neural network caused by TB40/E infection can be prevented by neutralizing antibodies (NAbs) that recognize different epitopes of the HCMV envelope pentamer complex (PC), a major target of HCMV-specific humoral immunity. These results demonstrate in a three-dimensional human cellular biosystem that HCMV can cause severe brain malformation and disrupt calcium signaling and neural network activity. This study also provides insights into the potential capacity of NAbs to mitigate brain defects resulted from congenital HCMV infection.
Project description:Human cytomegalovirus (hCMV) primo-infection, reinfection and/or reactivation is a major issue during pregnancy and affects 1% of live births in western countries, making hCMV the most frequently transmitted virus in utero. Despite the extensive research conducted so far, the pathophysiology of this congenital infection remains unclear. Recently, increasing evidence point out the role of small extracellular vesicles (sEVs) in cell-cell communication underlying the feto-placenta-maternal dialogue during pregnancy. In this study, we examined the impact of hCMV infection on the protein composition and function of placental sEVs. We observed that infection of placental cells led to an alteration of protein composition of their secreted sEVs, suggesting that placental sEVs may acquire a proviral phenotype. Functional studies performed on fetal recipient cells, notably neural stem cells, confirmed the ability of sEVs produced by infected cells to facilitate further infection of naive recipient cells. Altogether, our study demonstrates that placental sEVs are key players of hCMV pathophysiology during congenital infection, and may favor the transmission of the virus towards the fetus.