Biology subjects

Tidball, A. M.

Publications and source records attributed to Tidball, A. M..

6 recordsLinked to original sources

bioRxiv2026

High-Throughput Morphological Profiling of Patient-Derived Human Liver Organoids for Hepatotoxicity Hazard Identification and Compound Prioritization

Drug-induced liver injury (DILI) remains a major challenge in drug development because existing in vitro models poorly predict clinical hepatotoxicity. We coupled patient-derived human liver organoids (HLOs) with high-content imaging and automated machine learning (ML) to detect hepatotoxicity-associated phenotypes by morphological profiling. HLOs from six DILI patients and one healthy donor (n = 7) retained donor-specific baseline phenotypes in hepatocyte maturity, metabolism, and morphology. ML models trained per line on MAD-robustized, well-level-aggregated median features achieved compound-level AUROCs of 0.88 (95% CI 0.86-0.90) to 0.91 (95% CI 0.89-0.93) and balanced accuracies of 0.81-0.84 in distinguishing 54 hepatotoxicants from 20 non-hepatotoxic controls. Averaging predictions across all seven lines at an optimized threshold gave 94.4% sensitivity (95% CI 84.6-98.8%) and 85.0% specificity (95% CI 62.1-96.8%), substantially outperforming HLO cytotoxicity assays and cancer cell line profiling. Lines were moderately-to-highly concordant (Pearson r = 0.66-0.85), and benchmark concentration modeling revealed reproducible inter-line differences in phenotypic sensitivity (median 4.0-fold, several compounds exceeding 20-fold). Exposure anchoring of benchmark concentrations to human-equivalent plasma concentrations, using in vitro mass-balance dosimetry and curated plasma free fractions, placed organoid points of departure within 100-fold of clinical Cmax for 60 of 66 compounds (23-fold residual offset, 95% CI 12-45), and placed two of the three misclassified negative controls an order of magnitude above clinical Cmax. Sulindac injury signatures were significantly enhanced by TNF co-treatment, demonstrating capacity to detect pro-inflammatory-mediated DILI. This platform preserves inter-line variability while enabling reproducible, scalable multi-line consensus hazard classification; because compounds were tested over a common nominal concentration range, the readout defines hepatocellular hazard, which must be combined with exposure as an independent input to estimate clinical DILI risk.

pharmacology and toxicology↗

bioRxiv2025

Robust Production of Parvalbumin Cortical Interneurons and Fast-Spiking Neurons from Human Medial Ganglionic Eminence Organoids

The medial ganglionic eminence (MGE) gives rise to parvalbumin (PV)-and somatostatin (SST)-expressing cortical interneurons essential for regulating cortical excitability. Although PV interneurons are linked to various neurodevelopmental and neurodegenerative disorders, reliably generating them from human pluripotent stem cells (hPSCs) has been extremely challenging. We present a robust, reproducible protocol for generating single-rosette MGE organoids (MGEOs) from hPSCs. Transcriptomic analyses reveal that MGEOs exhibit MGE regional identity and faithfully model the developing human fetal MGE. As MGEOs mature, they generate abundant PV-expressing cortical interneurons, including putative basket and axoaxonic cells, at a scale not previously achieved in vitro. When fused with human cortical organoids (hCOs), these interneurons rapidly migrate into the hCOs, integrate into excitatory networks, and contribute to complex electrophysiological patterns and the emergence of large numbers of fast-spiking neurons. Using this model, we uncover a previously unreported migration deficit of MGE interneurons in a disease model of SLC6A1 developmental and epileptic encephalopathy, offering potential insights into the developmental contributions to epileptogenesis. MGEOs thus offer a powerful in vitro approach for probing human MGE-lineage cortical and subcortical GABAergic neuron development, modeling various neuropsychiatric disorders, and advancing cell-based therapies for neurodevelopmental and neurodegenerative disorders. HighlightsO_LIGeneration of subpallial organoids highly enriched for MGE lineages C_LIO_LIMGE organoids (MGEOs) robustly produce parvalbumin-expressing cortical interneurons C_LIO_LIComplex network activity and fast-spiking neurons are generated in assembloids C_LIO_LIImpaired interneuron migration in SLC6A1 knockout and patient-derived MGEOs C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/662594v2_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@1fce574org.highwire.dtl.DTLVardef@3c28d2org.highwire.dtl.DTLVardef@19bf737org.highwire.dtl.DTLVardef@834f2b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Genome-wide CRISPRi Screen in Human iNeurons to Identify Novel Focal Cortical Dysplasia Genes

Focal cortical dysplasia (FCD) is a common cause of focal epilepsy that typically results from brain mosaic mutations in the mTOR cell signaling pathway. To identify new FCD genes, we developed an in vitro CRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6 ribosomal protein (pS6). Using whole-genome (110,000 gRNAs) and candidate (129 gRNAs) libraries, we discovered 12 new genes that significantly increase pS6 levels. Interestingly, positive hits were enriched for brain-specific genes, highlighting the effectiveness of using human iPSC-derived induced neurons (iNeurons) in our screen. We investigated the signaling pathways of six candidate genes: LRRC4, EIF3A, TSN, HIP1, PIK3R3, and URI1. All six genes increased phosphorylation of S6. However, only two genes, PIK3R3 and HIP1, caused hyperphosphorylation more proximally in the AKT/mTOR/S6 signaling pathway. Importantly, these two genes have recently been found independently to be mutated in resected brain tissue from FCD patients, supporting the predictive validity of our screen. Knocking down each of the other four genes (LRRC4, EIF3A, TSN, and URI1) in iNeurons caused them to become resistant to the loss of growth factor signaling; without growth factor stimulation, pS6 levels were comparable to growth factor stimulated controls. Our data markedly expand the set of genes that are likely to regulate mTOR pathway signaling in neurons and provide additional targets for identifying somatic gene variants that cause FCD. Significance StatementFocal cortical dysplasia (FCD) is a common cause of focal epilepsy due to somatic variants in mTOR pathway genes in FCD brain tissue. Unbiased sequencing to identify novel FCD genes is challenging since these variants are often in a small subset of neurons. To overcome this challenge, we used an in vitro genetic screen in human neurons using an FCD biomarker, uncovering genes that increase neuronal mTOR signaling when their expression is lost. Two candidate genes were mutated in patient FCD brain tissues in a recent study, supporting a causative role for these genes in FCD. Our work suggests gene candidates for somatic variant analysis in FCD tissue and indicates added value for genetic screening in neurons over cell lines.

neuroscience↗

bioRxiv2023

A shared pathogenic mechanism for valproic acid and SHROOM3 knockout in a brain organoid model of neural tube defects

Neural tube defects (NTDs) including anencephaly and spina bifida are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or life-long severe complications (spina bifida). Despite hundreds of genetic mouse models having neural tube defect phenotypes, the genetics of human NTDs are poorly understood. Furthermore, pharmaceuticals such as antiseizure medications have been found clinically to increase the risk of NTDs when administered during pregnancy. Therefore, a model that recapitulates human neurodevelopment would be of immense benefit to understand the genetics underlying NTDs and identify teratogenic mechanisms. Using our self-organizing single rosette spheroid (SOSRS) brain organoid system, we have developed a high-throughput image analysis pipeline for evaluating SOSRS structure for NTD-like phenotypes. Similar to small molecule inhibition of apical constriction, the antiseizure medication valproic acid (VPA), a known cause of NTDs, increases the apical lumen size and apical cell surface area in a dose-responsive manner. This expansion was mimicked by GSK3{beta} and HDAC inhibitors; however, RNA sequencing suggests VPA does not inhibit GSK3{beta} at these concentrations. Knockout of SHROOM3, a well-known NTD-related gene, also caused expansion of the lumen as well as reduced f-actin polarization. The increased lumen sizes were caused by reduced cell apical constriction suggesting that impingement of this process is a shared mechanism for VPA treatment and SHROOM3-KO, two well-known causes of NTDs. Our system allows the rapid identification of NTD-like phenotypes for both compounds and genetic variants and should prove useful for understanding specific NTD mechanisms and predicting drug teratogenicity.

neuroscience↗

bioRxiv2022

Self-organizing Single-Rosette Brain Organoids from Human Pluripotent Stem Cells

The field of brain organoid research is complicated by morphological variability with multiple neural rosette structures per organoid. We have developed a new human brain organoid technique that generates self-organizing, single-rosette spheroids (SOSRS) with reproducible size, cortical-like lamination, and cell diversity. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells (hPSCs) that is patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D transition from neural plate to neural tube that occurs during neurodevelopment. The vast majority of monolayer fragments form spheres with a single central lumen and consistent growth rates. Over time, the SOSRS develop appropriately ordered lamination consistent with six cortical layers by immunocytochemistry and single cell RNA-sequencing. The reproducibility of this method has allowed us to demonstrate robust structural phenotypes arising from chemical teratogen exposure or when modeling a genetic neurodevelopmental epileptic disorder. This platform should advance studies of human cortical development, brain disorder mechanisms, and precision therapies. SUMMARY STATEMENTSimple procedure for generating reproducible single rosette cortical brain organoids used to identify robust structural phenotypes with neuroteratogen exposure and in a genetic neurodevelopmental disease model.

neuroscience↗

bioRxiv2020

Variant-specific changes in persistent or resurgent Na+ current in SCN8A-EIEE13 iPSC-derived neurons

Missense variants in the voltage-gated sodium channel (VGSC) gene, SCN8A, are linked to early-infantile epileptic encephalopathy type 13 (EIEE13). EIEE13 patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy (SUDEP). The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current (INa) properties. To investigate these mechanisms using a patient-specific model system, we generated induced pluripotent stem cells (iPSCs) from three patients with missense variants in SCN8A: p.R1872>L (P1); p.V1592>L (P2); and p.N1759>S (P3). Using small molecule differentiation into excitatory neurons, iPSC-derived neurons from all three patients displayed altered INa. P1 and P2 had elevated persistent INa, while P3 had increased resurgent INa compared to controls. Further analyses focused on one of the patients with increased persistent INa (P1) and the patient with increased resurgent INa (P3). Excitatory cortical neurons from both patients had prolonged action potential (AP) repolarization and shorter axon initial segment lengths compared to controls, the latter analyzed by immunostaining for ankyrin-G. Using doxycycline-inducible expression of the neuronal transcription factors Neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons (iNeurons), we investigated network activity and response to pharmacotherapies. Both patient neurons and iNeurons displayed similar abnormalities in AP repolarization. Patient iNeurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for EIEE patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent INa, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the AP firing threshold of patient-derived neurons to more depolarized potentials. Our results indicate that patient-specific neurons are useful for modeling EIEE13 and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific iPSC neuronal disease modeling offers a useful platform for discovering precision epilepsy therapies.

neuroscience↗