Resolving cell state in iPSC-derived human neural samples with multiplexed fluorescence imaging

Human induced pluripotent stem cell-derived (iPSC) neural cultures offer clinically relevant models of human diseases, including Amyotrophic Lateral Sclerosis, Alzheimer’s, and Autism Spectrum Disorder. In situ characterization of the spatial-temporal evolution of cell state in 3D culture and subsequent 2D dissociated culture models based on protein expression levels and localizations is essential to understanding neural cell differentiation, disease state phenotypes, and sample-to-sample variability. Here, we apply PRobe-based Imaging for Sequential Multiplexing (PRISM) to facilitate multiplexed imaging with facile, rapid exchange of imaging probes to analyze iPSC-derived cortical and motor neuron cultures that are relevant to psychiatric and neurodegenerative disease models, using over ten protein targets. Our approach permits analysis of cell differentiation, cell composition, and functional marker expression in complex stem-cell derived neural cultures. Furthermore, our approach is amenable to automation, offering in principle the ability to scale-up to dozens of protein targets and samples.

APOE2 mitigates disease-related phenotypes in an isogenic hiPSC-based model of Alzheimer’s disease

Genome-wide association studies (GWAS) have identified polymorphism in the Apolipoprotein E gene (APOE) to be the most prominent risk factor for Alzheimer’s disease (AD). Compared to individuals homozygous for the APOE3 variant, individuals with the APOE4 variant have a significantly elevated risk of AD. On the other hand, longitudinal studies have shown that the presence of the APOE2 variant reduces the lifetime risk of developing AD by 40 percent. While there has been significant research that has identified the risk-inducing effects of APOE4, the underlying mechanisms by which APOE2 influences AD onset and progression have not been extensively explored. In this study, we utilize an isogenic human induced pluripotent stem cell (hiPSC)-based system to demonstrate that conversion of APOE3 to APOE2 greatly reduced the production of amyloid-beta (Aβ) peptides in hiPSC-derived neural cultures. Mechanistically, analysis of pure populations of neurons and astrocytes derived from these neural cultures revealed that mitigating effects of APOE2 are mediated by cell autonomous and non-autonomous effects. In particular, we demonstrated the reduction in Aβ is potentially driven by a mechanism related to non-amyloidogenic processing of amyloid precursor protein (APP), suggesting a gain of the protective function of the APOE2 variant. Together, this study provides insights into the risk-modifying effects associated with the APOE2 allele and establishes a platform to probe the mechanisms by which APOE2 enhances neuroprotection against AD.

Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome

Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.

Comparison of Acute Effects of Neurotoxic Compounds on Network Activity in Human and Rodent Neural Cultures

Assessment of neuroactive effects of chemicals in cell-based assays remains challenging as complex functional tissue is required for biologically relevant readouts. Recent in vitro models using rodent primary neural cultures grown on multielectrode arrays (MEAs) allow quantitative measurements of neural network activity suitable for neurotoxicity screening. However, robust systems for testing effects on network function in human neural models are still lacking. The increasing number of differentiation protocols for generating neurons from human induced pluripotent stem cells (hiPSCs) holds great potential to overcome the unavailability of human primary tissue and expedite cell-based assays. Yet, the variability in neuronal activity, prolonged ontogeny and rather immature stage of most neuronal cells derived by standard differentiation techniques greatly limit their utility for screening neurotoxic effects on human neural networks. Here, we used excitatory and inhibitory neurons, separately generated by direct reprogramming from hiPSCs, together with primary human astrocytes to establish highly functional cultures with defined cell ratios. Such neuron/glia co-cultures exhibited pronounced neuronal activity and robust formation of synchronized network activity on MEAs, albeit with noticeable delay compared to primary rat cortical cultures. We further investigated acute changes of network activity in human neuron/glia co-cultures and rat primary cortical cultures in response to compounds with known adverse neuroactive effects, including GABAA receptor antagonists and multiple pesticides. Importantly, we observed largely corresponding concentration-dependent effects on multiple neural network activity metrics using both neural culture types. These results demonstrate the utility of directly converted neuronal cells from hiPSCs for functional neurotoxicity screening of environmental chemicals.

Modelling Lyssavirus Infections in Human Stem Cell-Derived Neural Cultures

Rabies is a zoonotic neurological infection caused by lyssavirus that continues to result in devastating loss of human life. Many aspects of rabies pathogenesis in human neurons are not well understood. Lack of appropriate ex-vivo models for studying rabies infection in human neurons has contributed to this knowledge gap. In this study, we utilize advances in stem cell technology to characterize rabies infection in human stem cell-derived neurons. We show key cellular features of rabies infection in our human neural cultures, including upregulation of inflammatory chemokines, lack of neuronal apoptosis, and axonal transmission of viruses in neuronal networks. In addition, we highlight specific differences in cellular pathogenesis between laboratory-adapted and field strain lyssavirus. This study therefore defines the first stem cell-derived ex-vivo model system to study rabies pathogenesis in human neurons. This new model system demonstrates the potential for enabling an increased understanding of molecular mechanisms in human rabies, which could lead to improved control methods.

Molecular correlates of topiramate and GRIK1 rs2832407 genotype in pluripotent stem cell-derived neural cultures

There is growing evidence that the anticonvulsant topiramate is efficacious in reducing alcohol consumption. Further, an intronic single nucleotide polymorphism (rs2832407, C ➔ A) in the GRIK1 gene, which encodes the GluK1 subunit of the excitatory kainate receptor, predicted topiramate’s effectiveness in reducing heavy drinking in a clinical trial. In the current study, we differentiated a total of 22 induced pluripotent stem cell (iPSCs) lines characterized by GRIK1 rs2832407 genotype (10 A/A and 12 C/C) into forebrain-lineage neural cultures to explore molecular correlates of GRIK1 genotype that may relate to topiramate’s ability to reduce drinking. Our differentiation protocol yielded mixed neural cultures enriched for glutamatergic neurons. Characterization of the GRIK1 locus revealed no effect of rs2832407 genotype on GRIK1 isoform mRNA expression, however a significant difference was observed on GRIK1 antisense-2, with higher expression in C/C neural cultures. Differential effects of acute exposure to 5 μM topiramate were observed on the frequency of spontaneous synaptic activity in A/A vs. C/C neurons, with a smaller reduction in excitatory event frequency and a greater reduction in inhibitory event frequency observed in C/C donor neurons. This work highlights the use of iPSC technologies to study pharmacogenetic treatment effects in psychiatric disorders and furthers our understanding of the molecular effects of topiramate exposure in human neural cells.

Exosomes regulate neurogenesis and circuit assembly

Exosomes are thought to be released by all cells in the body and to be involved in intercellular communication. We tested whether neural exosomes can regulate the development of neural circuits. We show that exosome treatment increases proliferation in developing neural cultures and in vivo in dentate gyrus of P4 mouse brain. We compared the protein cargo and signaling bioactivity of exosomes released by hiPSC-derived neural cultures lacking MECP2, a model of the neurodevelopmental disorder Rett syndrome, with exosomes released by isogenic rescue control neural cultures. Quantitative proteomic analysis indicates that control exosomes contain multiple functional signaling networks known to be important for neuronal circuit development. Treating MECP2-knockdown human primary neural cultures with control exosomes rescues deficits in neuronal proliferation, differentiation, synaptogenesis, and synchronized firing, whereas exosomes from MECP2-deficient hiPSC neural cultures lack this capability. These data indicate that exosomes carry signaling information required to regulate neural circuit development.