Human brain organoids as a model to study HCMV-induced neuropathogenesis and evaluate the activity of antiviral compounds

Introduction:Human cytomegalovirus (HCMV) belongs to betaherpesvirus subfamily. It is a ubiquitous pathogen that can establish lifelong latency in the human host. People without an adequate immune response may suffer from HCMV-related diseases, among which one of the most widespread is the vertical transmission in utero, from the mother to the developing fetus, i.e. congenital CMV infection (cCMV). cCMV represents a common cause of neurodevelopmental defects and can lead to CNS damage and neurodevelopmental deficits. Despite this infection being very common, its induced neuropathogenesis remains unclear. Moreover, no effective therapy has currently proven to prevent or treat this congenital infection. The main reason of this delay can be addressed to the lack of suitable experimental models, due to the strict species-specificity of the pathogen and to the complexity to faithfully reproduce the developing human brain. Aim of the study:The project aimed to optimize a 3D platform of cerebral organoids that can recapitulate the early-developing human brain and that can be exploited to study the effects of HCMV infection, comparing those effects with the ones detected upon drug treatment. The consequences of the infection were evaluated starting from a transcriptomic analysis of mock- and HCMV-infected organoids, validating the resulting gene in 2D-systems by quantitative PCR (qPCR). Then organoids were investigated for their susceptibility to HCMV and the viral diffusion in various phases of their maturation, evaluating the effects of antiviral drugs (ganciclovir, letermovir, nitazoxanide and OZ418) in reducing the infection by confocal microscopy. At the end, potential neural markers were evaluated at different maturation stages of the infected organoids by qPCR. Materials and methods:3D cerebral organoids were generated starting from H9 human embryonic stem cells (hESCs) following a protocol designed by Lancaster and Knoblich (2014), with minor modifications. Organoids were infected with HCMV TB40/E clinical strain expressing the fluorescent fusion protein pp65-YFP, with a multiplicity of infection (MOI) of 0.1 PFU/cell, and RNAseq analysis was performed. Resulting dysregulated genes were validated on 2D in vitro systems of neural progenitors cells. Brain organoids at different stages of maturation were then infected and treated with fixed concentrations of ganciclovir, letermovir, nitazoxanide and OZ418 for the remaining period of the experiments. The fluorescence signal was then monitored by confocal microscopy to assess the state of the infection at different days post infection (dpi), with and without the drug treatments. Additionally, organoid sections were obtained and stained for neural-specific markers. Real-time qPCR was performed on neural stem cells, neurepithelial stem cells and on organoid-derived cDNA to detect and quantify the expression of viral and neural-specific genes, together with markers of pluripotency and differentiation. Results:Cerebral organoids have been correctly generated and, when matured, presented actively replicating pluripotent neural progenitor cells (NPCs) organized in neural rosettes. NPCs were surrounded by regions full of immature neural cells, interspersed with terminally differentiated neurons. By transcriptomic analysis, 300 genes were found upregulated by the infection in 30 days-infected organoids, and 2 out of 4 genes resulted differentially expressed also in 2D-systems. Mature tissues, starting from day 20, were efficiently infected with pp65-YFP, showing a widespread and persistent fluorescence signal, with an efficient activity of antiviral drugs in counteracting viral replication. Finally, two potential markers tested on organoids by qPCR at different stages, resulted downregulated in their most mature differentiation stage. Conclusions: This study confirmed the reliability of this model to correctly recapitulate the human developing brain in its initial stages.