Pluripotent stem cell-derived neural progenitor cells can be used to model effects of IL-6 on human neurodevelopment.

Maternal immune activation (MIA) increases the risks for neurodevelopmental disorders in offspring through inflammatory cytokines, including interleukin-6 (IL-6). We therefore aimed to establish a human two-dimensional (2D) in vitro neural model to investigate the effects of IL-6 exposure on neurodevelopment. IL-6 signal transduction requires two receptors: interleukin-6 signal transducer (IL6ST) and interleukin-6 receptor (IL6R). Prenatally, neural cells lack IL6R, and hence cannot elicit cis IL-6 signaling, but IL6R can be provided by microglia in trans. We demonstrate here that an immortalized human neural progenitor cell (NPC) line, ReNCell CX, expresses IL6ST and elicits both cis and trans IL-6 signaling, limiting its use as a model of MIA. In contrast, induced pluripotent stem cell (iPSC)-derived NPCs only activate the IL-6 cascade in trans. Activation of the trans IL-6 cascade did not result in increased proliferation of iPSC-derived NPCs or ReNCell CX, as has been demonstrated in animal models. iPSC-derived NPCs upregulated NR2F1 expression in response to IL-6 signaling in line with analogous experiments in organoids. Thus, iPSC-derived NPCs can be used to model gene expression changes in response to MIA in 2D cultures.

Human brain organoid model of maternal immune activation identifies radial glia cells as selectively vulnerable.

Maternal immune activation (MIA) during critical windows of gestation is correlated with long-term neurodevelopmental deficits in the offspring, including increased risk for autism spectrum disorder (ASD) in humans. Interleukin 6 (IL-6) derived from the gestational parent is one of the major molecular mediators by which MIA alters the developing brain. In this study, we establish a human three-dimensional (3D) in vitro model of MIA by treating induced pluripotent stem cell-derived dorsal forebrain organoids with a constitutively active form of IL-6, Hyper-IL-6. We validate our model by showing that dorsal forebrain organoids express the molecular machinery necessary for responding to Hyper-IL-6 and activate STAT signaling upon Hyper-IL-6 treatment. RNA sequencing analysis reveals the upregulation of major histocompatibility complex class I (MHCI) genes in response to Hyper-IL-6 exposure, which have been implicated with ASD. We find a small increase in the proportion of radial glia cells after Hyper-IL-6 treatment through immunohistochemistry and single-cell RNA-sequencing. We further show that radial glia cells are the cell type with the highest number of differentially expressed genes, and Hyper-IL-6 treatment leads to the downregulation of genes related to protein translation in line with a mouse model of MIA. Additionally, we identify differentially expressed genes not found in mouse models of MIA, which might drive species-specific responses to MIA. Finally, we show abnormal cortical layering as a long-term consequence of Hyper-IL-6 treatment. In summary, we establish a human 3D model of MIA, which can be used to study the cellular and molecular mechanisms underlying the increased risk for developing disorders such as ASD.

Tissue clearing may alter emission and absorption properties of common fluorophores.

In recent years, 3D cell culture has been gaining a more widespread following across many fields of biology. Tissue clearing enables optical analysis of intact 3D samples and investigation of molecular and structural mechanisms by homogenizing the refractive indices of tissues to make them nearly transparent. Here, we describe and quantify that common clearing solutions including benzyl alcohol/benzyl benzoate (BABB), PEG-associated solvent system (PEGASOS), immunolabeling-enabled imaging of solvent-cleared organs (iDISCO), clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and ScaleS4 alter the emission spectra of Alexa Fluor fluorophores and fluorescent dyes. Clearing modifies not only the emitted light intensity but also alters the absorption and emission peaks, at times to several tens of nanometers. The resulting shifts depend on the interplay of solvent, fluorophore, and the presence of cells. For biological applications, this increases the risk for unexpected channel crosstalk, as filter sets are usually not optimized for altered fluorophore emission spectra in clearing solutions. This becomes especially problematic in high throughput/high content campaigns, which often rely on multiband excitation to increase acquisition speed. Consequently, researchers relying on clearing in quantitative multiband excitation experiments should crosscheck their fluorescent signal after clearing in order to inform the proper selection of filter sets and fluorophores for analysis.

Cell-Type-Specific High Throughput Toxicity Testing in Human Midbrain Organoids.

Toxicity testing is a crucial step in the development and approval of chemical compounds for human contact and consumption. However, existing model systems often fall short in their prediction of human toxicity in vivo because they may not sufficiently recapitulate human physiology. The complexity of three-dimensional (3D) human organ-like cell culture systems ("organoids") can generate potentially more relevant models of human physiology and disease, including toxicity predictions. However, so far, the inherent biological heterogeneity and cumbersome generation and analysis of organoids has rendered efficient, unbiased, high throughput evaluation of toxic effects in these systems challenging. Recent advances in both standardization and quantitative fluorescent imaging enabled us to dissect the toxicities of compound exposure to separate cellular subpopulations within human organoids at the single-cell level in a framework that is compatible with high throughput approaches. Screening a library of 84 compounds in standardized human automated midbrain organoids (AMOs) generated from two independent cell lines correctly recognized known nigrostriatal toxicants. This approach further identified the flame retardant 3,3',5,5'-tetrabromobisphenol A (TBBPA) as a selective toxicant for dopaminergic neurons in the context of human midbrain-like tissues for the first time. Results were verified with high reproducibility in more detailed dose-response experiments. Further, we demonstrate higher sensitivity in 3D AMOs than in 2D cultures to the known neurotoxic effects of the pesticide lindane. Overall, the automated nature of our workflow is freely scalable and demonstrates the feasibility of quantitatively assessing cell-type-specific toxicity in human organoids in vitro.

A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids.

Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates ('organoids') or 3D structures with less physiological relevance ('spheroids'). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.

In 1907, the American zoologist Ross Granville Harrison developed the first technique to artificially grow animal cells outside the body in a liquid medium. Cells are still grown in much the same way in modern laboratories: a single layer of cells is placed in a warm incubator with nutrient-rich broth. These cell layers are often used to test new drugs, but they cannot recapitulate the complexity of a real organ made from multiple cell types within a living, breathing human body. Growing three-dimensional miniature organs or 'organoids' that behave in a similar way to real organs is the next step towards creating better platforms for drug screening, but there are several difficulties inherent to this process. For one thing, it is hard to recreate the multitude of cell types that make up an organ. For another, the cells that do grow often fail to connect and communicate with each other in biologically realistic ways. It is also tough to grow a large number of organoids that all behave in the same way, making it hard to know whether a particular drug works or whether it is just being tested on a 'good' organoid. Renner et al. have been able to overcome these issues by using robotic technology to create thousands of identical, mid-brain organoids from human cells in the lab. The robots perform a series of precisely controlled tasks ­ including dispensing the initial cells into wells, feeding organoids as they grow and testing them at different stages of development. These mini-brains, which are the size of the head of a pin, mimic the part of the brain where Parkinson's disease first manifests. They can be used to test new drugs for Parkinson's, and to better understand the biology of the brain. Perhaps more importantly, other types of organoids can be created using the same technique to model diseases that affect other areas of the brain, or other organs altogether. For example, Renner et al. also generated forebrain organoids using an automated approach for both generation and analysis. This research, which shows that organoids can be grown and tested in a fully automated, reproducible and scalable way, creates a platform to quickly, cheaply and easily test thousands of drugs for Parkinson's and other difficult-to-treat diseases in a human setting. This approach has the potential to reduce research waste by increasing the chances that a drug that works in the lab will also ultimately work in a patient; and reduce animal experiments, as drugs that do not work in human tissues will not proceed to animal testing.