Phenotypic Screening of Prospective Analgesics Among FDA-Approved Compounds using an iPSC-Based Model of Acute and Chronic Inflammatory Nociception.

Classical target-based drug screening is low-throughput, largely subjective, and costly. Phenotypic screening based on in vitro models is increasingly being used to identify candidate compounds that modulate complex cell/tissue functions. Chronic inflammatory nociception, and subsequent chronic pain conditions, affect peripheral sensory neuron activity (e.g., firing of action potentials) through myriad pathways, and remain unaddressed in regard to effective, non-addictive management/treatment options. Here, a chronic inflammatory nociception model is demonstrated based on induced pluripotent stem cell (iPSC) sensory neurons and glia, co-cultured on microelectrode arrays (MEAs). iPSC sensory co-cultures exhibit coordinated spontaneous extracellular action potential (EAP) firing, reaching a stable baseline after ≈27 days in vitro (DIV). Spontaneous and evoked EAP metrics are significantly modulated by 24-h incubation with tumor necrosis factor-alpha (TNF-α), representing an inflammatory phenotype. Compared with positive controls (lidocaine), this model is identified as an "excellent" stand-alone assay based on a modified Z' assay quality metric. This model is then used to screen 15 cherry-picked, off-label, Food and Drug Administration (FDA)-approved compounds; 10 of 15 are identified as "hits". Both hits and "misses" are discussed in turn. In total, this data suggests that iPSC sensory co-cultures on MEAs may represent a moderate-to-high-throughput assay for drug discovery targeting inflammatory nociception.

A 3D In Vitro Cortical Tissue Model Based on Dense Collagen to Study the Effects of Gamma Radiation on Neuronal Function.

Studies on gamma radiation-induced injury have long been focused on hematopoietic, gastrointestinal, and cardiovascular systems, yet little is known about the effects of gamma radiation on the function of human cortical tissue. The challenge in studying radiation-induced cortical injury is, in part, due to a lack of human tissue models and physiologically relevant readouts. Here, a physiologically relevant 3D collagen-based cortical tissue model (CTM) is developed for studying the functional response of human iPSC-derived neurons and astrocytes to a sub-lethal radiation exposure (5 Gy). Cytotoxicity, DNA damage, morphology, and extracellular electrophysiology are quantified. It is reported that 5 Gy exposure significantly increases cytotoxicity, DNA damage, and astrocyte reactivity while significantly decreasing neurite length and neuronal network activity. Additionally, it is found that clinically deployed radioprotectant amifostine ameliorates the DNA damage, cytotoxicity, and astrocyte reactivity. The CTM provides a critical experimental platform to understand cell-level mechanisms by which gamma radiation (GR) affects human cortical tissue and to screen prospective radioprotectant compounds.

Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro.

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Underscoring the effect of swab type, workflow, and positive sample order on swab pooling for COVID-19 surveillance testing.

Sample pooling is a promising strategy to facilitate COVID-19 surveillance testing for a larger population in comparison to individual single testing due to resource and time constraints. Increased surveillance testing capacity will reduce the likelihood of outbreaks as the general population is returning to work, school, and other gatherings. We have analyzed the impact of three variables on the effectiveness of pooling test samples: swab type, workflow, and positive sample order. We investigated the performance of several commercially available swabs (Steripack polyester flocked, Puritan nylon flocked, Puritan foam) in comparison to a new injected molded design (Yukon). The bench-top performance of collection swab was conducted with a previously developed anterior nasal cavity tissue model, based on a silk-glycerol sponge to mimic soft tissue mechanics and saturated with a physiologically relevant synthetic nasal fluid spiked with heat-inactivated SARS-CoV-2. Overall, we demonstrated statistically significant differences in performance across the different swab types. A characterization of individual swab uptake (gravimetric analysis) and FITC microparticle release suggests that differences in absorbance and retention drive the observed differences in Ct of the pooled samples. We also proposed two distinct pooling workflows to encompass different community collection modes and analyzed the difference in resulting positive pools as an effect of workflow, swab type, and positive sample order. Overall, swab types with lower volume retention resulted in reduced false negative occurrence, also observed for collection workflows with limited incubation times. Concurrently, positive sample order did have a significant impact on pooling test outcome, particularly in the case of swab type with great volume retention. We demonstrated that the variables investigated here affect the results of pooled COVID-19 testing, and therefore should be considered while designing pooled surveillance testing.

A Complement C3-Specific Nanobody for Modulation of the Alternative Cascade Identifies the C-Terminal Domain of C3b as Functional in C5 Convertase Activity.

The complement system is an intricate cascade of the innate immune system and plays a key role in microbial defense, inflammation, organ development, and tissue regeneration. There is increasing interest in developing complement regulatory and inhibitory agents to treat complement dysfunction. In this study, we describe the nanobody hC3Nb3, which is specific for the C-terminal C345c domain of human and mouse complement component C3/C3b/C3c and potently inhibits C3 cleavage by the alternative pathway. A high-resolution structure of the hC3Nb3-C345c complex explains how the nanobody blocks proconvertase assembly. Surprisingly, although the nanobody does not affect classical pathway-mediated C3 cleavage, hC3Nb3 inhibits classical pathway-driven hemolysis, suggesting that the C-terminal domain of C3b has an important function in classical pathway C5 convertase activity. The hC3Nb3 nanobody binds C3 with low nanomolar affinity in an SDS-resistant complex, and the nanobody is demonstrated to be a powerful reagent for C3 detection in immunohistochemistry and flow cytometry. Overall, the hC3Nb3 nanobody represents a potent inhibitor of both the alternative pathway and the terminal pathway, with possible applications in complement research, diagnostics, and therapeutics.