Project description:This study investigated the viral mechanisms of fetal neural malformation using in vitro human cerebral organoids to model congenital CMV infection
Project description:Human cytomegalovirus (HCMV) is a betaherpesvirus that can cause severe birth defects including vision and hearing loss, microcephaly, and seizures. Currently, no approved treatment options exist for in utero infections. Here, we aimed to determine the impact of HCMV infection on the transcriptome of developing neurons in an organoid model system. Cell populations isolated from organoids based on a marker for infection and transcriptomes were defined. We uncovered downregulation in key cortical, neurodevelopmental, and functional gene pathways which occurred regardless of the degree of infection. To test the contributions of specific HCMV immediate early proteins known to disrupt neural differentiation, we infected NPCs using a recombinant virus harboring a destabilization domain. Despite suppressing their expression, HCMV-mediated transcriptional downregulation still occurred. Together, our studies have revealed that HCMV infection causes a profound downregulation of neurodevelopmental genes and suggest a role for other viral factors in this process.
Project description:We report the application of bulk RNA sequencing on cerebral organoids infected with HCMV TB40eGFP virus or uninfected, within the infected groups the organoids were sorted into 3 distinct populations prior to sequencing based on GFP expression as a proxy for level of infection. These populations have been denoted GFP (+), GFP Inter, GFP (-), for each sorted population 3 organoids of the same line and from the same differentiation were combined to ensure sufficient cell numbers for bulk sequencing analysis.
Project description:Recombinant Towne CMV expressing luciferase under the control of CMV-DNA polymerase (POL) or the late pp28 (UL99) promoters were evaluated for potential application in high-throughput screening of anti-viral compounds. POL-and pp28-luciferase displayed maximal expression 48 and 72 hours post infection, respectively. The pp28-luciferase virus achieved a wider dynamic range of luciferase expression (6-7 logs) and was selected for testing of inhibition by five anti-viral compounds. Luciferase expression highly correlated with plaque reduction and real-time PCR. The pp28-luciferase reporter system is rapid, reproducible, and highly sensitive. It may be applied to screening of novel anti-CMV compounds.
Project description:The role of human cytomegalovirus (HCMV)-specific T-cell responses in breast milk of HCMV-seropositive mothers is not well defined. In these studies, we demonstrate that the frequency of cytomegalovirus (CMV)-pp65-specific T-cell responses in peripheral blood mononuclear cells (PBMCs) and breast milk cells (BMCs) is increased for CD8+ T cells in both sample sources when compared with CD4+ T cells. The frequency of pp55-specific CD8 T cells producing interferon γ (IFN-γ) alone or dual IFN-γ/granzyme rB producers is increased in breast milk compared with PBMCs. Lastly, we observed a positive correlation between breast milk viral load and the CD8 pp65-specific response, suggesting that local virus replication drives antigen-specific CD8 T cells into the breast.
Project description:Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.
Project description:We have developed a cancer model of gliomas in human cerebral organoids that allows direct observation of tumor initiation as well as continuous microscopic observations. We used CRISPR/Cas9 technology to target an HRasG12V-IRES-tdTomato construct by homologous recombination into the TP53 locus. Results show that transformed cells rapidly become invasive and destroy surrounding organoid structures, overwhelming the entire organoid. Tumor cells in the organoids can be orthotopically xenografted into immunodeficient NOD/SCID IL2RG-/- animals, exhibiting an invasive phenotype. Organoid-generated putative tumor cells show gene expression profiles consistent with mesenchymal subtype human glioblastoma. We further demonstrate that human-organoid-derived tumor cell lines or primary human-patient-derived glioblastoma cell lines can be transplanted into human cerebral organoids to establish invasive tumor-like structures. Our results show potential for the use of organoids as a platform to test human cancer phenotypes that recapitulate key aspects of malignancy.
Project description:Brain organoids recapitulate in vitro the specific stages of in vivo human brain development, thus offering an innovative tool by which to model human neurodevelopmental disease. We review here how brain organoids have been used to study neurodevelopmental disease and consider their potential for both technological advancement and therapeutic development.
Project description:Although congenital infection by human cytomegalovirus (HCMV) is well recognized as a leading cause of neurodevelopmental defects, HCMV neuropathogenesis remains poorly understood. A major challenge for investigating HCMV-induced abnormal brain development is the strict CMV species specificity, which prevents the use of animal models to directly study brain defects caused by HCMV. We show that infection of human-induced pluripotent-stem-cell-derived brain organoids by a "clinical-like" HCMV strain results in reduced brain organoid growth, impaired formation of cortical layers, and abnormal calcium signaling and neural network activity. Moreover, we show that the impeded brain organoid development caused by HCMV can be prevented by neutralizing antibodies (NAbs) that recognize the HCMV pentamer complex. These results demonstrate in a three-dimensional cellular biosystem that HCMV can impair the development and function of the human brain and provide insights into the potential capacity of NAbs to mitigate brain defects resulted from HCMV infection.