Biology subjects

Zhang, C. J.

Publications and source records attributed to Zhang, C. J..

11 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↗

bioRxiv2026

Failure to release from the ER translocon induces 60S ribosome degradation

Ribosome malfunction has toxic consequences, but how mammalian cells select ribosomal subunits for degradation remains poorly understood. Here, we develop a pulse-chase CRISPR screening strategy to identify regulators of ribosome turnover and uncover that loss of protein UFMylation promotes ribosome degradation. Normal UFMylation releases the 60S subunit from the endoplasmic reticulum (ER) translocon. Without UFMylation, single-molecule tracking and optogenetic proximity labeling reveal accumulation of ER-associated 60S subunits and their specific degradation. This translocon-associated large subunit degradation (TLSD) is independent of the lysosome and, in our human cell model, is only active in nondividing, contact-inhibited cells. Degradation requires the poorly characterized protein R3HDM4, which we term TLSD1. Upon UFMylation loss, TLSD1 redistributes to the ER and promotes 60S degradation. These findings identify a cell-state-dependent pathway for clearing ER-retained 60S subunits. We propose translocon release acts as a checkpoint for ribosome function, explaining TLSD1's genetic links to neuronal health and erythropoiesis.

cell biology↗

bioRxiv2026

Suspension-Based Human Esophagoids Recapitulate WNT2B-Dependent Regulation of Esophageal Basal Progenitors

Background & AimsThe human esophagus undergoes a tightly regulated developmental program, transitioning from a simple columnar epithelium in early development to a mature stratified squamous tissue essential for adult barrier function. Here, we constructed a developmental cell atlas spanning early development to adulthood and leveraged it to generate physiologically relevant in vitro models. MethodsWe utilized single-cell RNA sequencing and spatial multiplex proteomics of human esophageal tissue from early development through adulthood. We established a feeder-supported 2D culture system and a Matrigel-free, suspension-based 3D esophagoid model in a 96-well format. To interrogate WNT2B function, we analyzed patient tissue harboring WNT2B loss-of-function mutations and performed WNT inhibition in esophagoids. ResultsSequencing profiling identified stage-specific epithelial populations: multiciliated and GPC3 basal cells were unique to early development; KRT14 basal and CRNN luminal cells were adult-specific; and COL17A1, LY6D, and KRT4 populations were shared across stages. Spatially organized WNT2B, KIT, and VWC2 mesenchymal subtypes were identified. The 2D system preserved both epithelial and mesenchymal compartments with transcriptional fidelity. Esophagoids exhibited basal-to-luminal stratification, mesenchymal compartmentalization, and required stromal interactions for formation. WNT2B repressed self-renewal of TP63 basal progenitors and inhibited proliferation, confirmed by pharmacologic inhibition of WNT in the in vitro esophagoids. ConclusionsWe present a stage-resolved atlas of human esophageal development and a scalable esophagoid platform recapitulating esophageal architecture. WNT2B regulates progenitor dynamics by restraining basal cell self-renewal. Esophagoids provide a physiologically relevant system for modeling esophageal development and disease. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/733451v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1af5743org.highwire.dtl.DTLVardef@8a061dorg.highwire.dtl.DTLVardef@1975c3forg.highwire.dtl.DTLVardef@292ce9_HPS_FORMAT_FIGEXP M_FIG C_FIG Key Findings and ImplicationsO_LIDevelopmental Atlas: The study presents a comprehensive transcriptional and structural atlas of the human esophageal epithelium, identifying conserved and stage-specific epithelial populations from early development to adulthood. Notably, stage-specific gene expression of multiciliated and GPC3 basal cells were unique to early development, while KRT14 basal and CRNN luminal cells were adult specific, with COL17A1+ (basal), LY6D+ (epibasal), and KRT4+ (middle), shared at all stages. C_LIO_LIMesenchymal Diversity: Spatial and transcriptional profiling revealed distinct mesenchymal subtypes, including WNT2B, KIT, and VWC2 populations, which are spatially organized and contribute to epithelial-mesenchymal signaling. These findings reinforce the role of stromal-epithelial interactions in esophageal development. C_LIO_LI2D Esophagus Cell Culture System: A feeder-supported 2D cell culture system was developed that retains both epithelial and mesenchymal populations, preserving transcriptional fidelity and enabling long-term expansion for mechanistic studies. C_LIO_LI3D Esophagoid Model: A suspension-based 3D organoid system was optimized using a 96-well format, enabling high-throughput generation of esophagoids with robust epithelial stratification and mesenchymal compartmentalization. These organoids recapitulate key features of the human esophagus, including basal-to-luminal organization, and require stromal interactions for formation. C_LIO_LIFunctional Role of WNT2B in esophagus development: Both in vivo and in vitro analyses demonstrated that WNT2B regulates epithelial progenitor dynamics and tissue architecture by repressing self-renewal of basally localized TP63+ cells and inhibiting proliferation. Loss-of-function models and WNT pathway modulation confirmed its role in epithelial-mesenchymal crosstalk and organoid integrity. C_LI

developmental biology↗

bioRxiv2024

LUMIC: Latent diffUsion for Multiplexed Images of Cells

The rapid advancement of high-content, single-cell technologies like robotic confocal microscopy with multiplexed dyes (morphological profiling) can be leveraged to reveal fundamental biology, ranging from microbial and abiotic stress to organ development. Specifically, heterogeneous cell systems can be perturbed genetically or with chemical treatments to allow for inference of causal mechanisms. An exciting strategy to navigate the high-dimensional space of possible perturbation and cell type combinations is to use generative models as priors to anticipate high-content outcomes in order to design informative experiments. Towards this goal, we present the Latent diffUsion for Multiplexed Images of Cells (LUMIC) framework that can generate high quality and high fidelity images of cells. LUMIC combines diffusion models with DINO (self-Distillation with NO labels), a vision-transformer based, self-supervised method that can be trained on images to learn feature embeddings, and HGraph2Graph, a hierarchical graph encoder-decoder to represent chemicals. To demonstrate the ability of LUMIC to generalize across cell lines and treatments, we apply it to a dataset of[~] 27,000 images of two cell lines treated with 306 chemicals and stained with three dyes from the JUMP Pilot dataset and a newly-generated dataset of[~] 3,000 images of five cell lines treated with 61 chemicals and stained with three dyes. To quantify prediction quality, we evaluate the DINO embeddings, Kernel Inception Distance (KID) score, and recovery of morphological feature distributions. LUMIC significantly outperforms previous methods and generates realistic out-of-sample images of cells across unseen compounds and cell types.

molecular biology↗

bioRxiv2023

A High-Throughput Microphysiological Liver Chip System to Model Direct and Idiosyncratic Drug-Induced Liver Injury Using Human Liver Organoids

1.ObjectiveDrug-induced liver injury (DILI) is a major failure mode in pharmaceutical development. This study aims to address the limitations of existing preclinical models by introducing a high-throughput, microfluidic liver-on-a-chip system, termed as "Curio Barrier Liver Chips," seeded with human liver organoids to enable metabolic and phenotypic morphologic characterization. MethodsCurio Barrier liver chips, fabricated in an 8x2 well configuration, were utilized to establish 3D liver organoid cultures. Human-induced pluripotent stem cells (iPSCs) were differentiated into hepatic-like organoids (HLOs), and their viability, liver-specific functions, and pharmacological responses were assessed over 28 days. ResultsThe Curiochips successfully maintained liver physiology and function, showing strong albumin secretion and cytochrome P450 (CYP) activities through 28 days. Unlike traditional models requiring millimolar drug concentrations to detect hepatotoxicity, this platform showed increased sensitivity for APAP and FIAU at micromolar concentrations. In situ differentiation of foregut spheroids to liver organoids was also achieved, further simplifying the establishment of liver chips. Furthermore, the chips demonstrated viability, function and DILI responsiveness for 28 days making this an improved model for studying idiosyncratic DILI with prolonged drug exposure and high-throughput capabilities compared to other available systems or primary human hepatocytes. ConclusionsThe Curiochips offer an advanced, miniaturized in vitro model for early-stage drug development and a sensitive, responsive and cost-effective means to detect direct hepatotoxicity. The high-throughput capability, coupled with robust functionality and pharmacological responses make it a promising tool for improving the prediction and understanding of DILI mechanisms in general and those that required prolonged drug exposure. The model also opens new avenues for research in other chronic liver diseases.

pharmacology and toxicology↗

bioRxiv2023

Opposing roles for TGFbeta and BMP-signaling during nascent alveolar differentiation in the developing human lung

Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGF{beta}- and BMP-signaling, where inhibition of TGF{beta}- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro. AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGF{beta}-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGF{beta}- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.

developmental biology↗

bioRxiv2023

Screening in serum-derived medium reveals differential response to compounds targeting metabolism

INTRODUCTION INTRODUCTION RESULTS DISCUSSION KEY RESOURCES TABLE CONTACT FOR REAGENT AND... EXPERIMENTAL MODEL AND SUBJECT... METHOD DETAILS QUANTIFICATION AND STATISTICAL... Data resources DATA AND SOFTWARE AVAILABILITY AUTHOR CONTRIBUTIONS DECLARATION OF INTERESTS REFERENCES Studies of cancer cells in standard culture conditions have long been used as a tractable tool for drug discovery. Cell culture provides unparalleled experimental flexibility, scalability, and low cost to identify and understand the response to cancer therapeutics; however, drug responses in culture are not always predictive of drug response in animal models or in patients1 ...

cancer biology↗

bioRxiv2022

In Vitro Evaluation and Mitigation of Niclosamide Liabilities as a COVID-19 Treatment.

Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including B.1.1.7. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future. ImportanceThere is still an urgent need for effective anti-SARS-CoV-2 therapeutics due to waning vaccine efficacy, the emergence of variants of concern, and limited efficacy of existing antivirals. One potential therapeutic option is niclosamide, an FDA approved anthelmintic compound that has shown promising anti-SARS-CoV-2 activity in cell-based assays. Unfortunately, there are significant barriers for the clinical utility of niclosamide as a COVID-19 therapeutic. Our work emphasizes these limitations by showing that niclosamide has high cytotoxicity at antiviral concentrations, variable potency against variants of concern, and significant polypharmacology as a result of its activity as a nonspecific protonophore. Some of these clinical limitations can be mitigated, however, through structural modifications to the niclosamide scaffold, which we demonstrate through a preliminary structure activity relationship analysis. Overall, we show that niclosamide is not a suitable candidate for the treatment of COVID-19, but that structural analogs with improved drug properties may have higher clinical-translational potential.

microbiology↗

bioRxiv2021

A Multi-Omics Human Liver Organoid Screening Platform for DILI Risk Prediction

Background and AimsDrug-induced liver injury (DILI), both intrinsic and idiosyncratic, causes frequent morbidity, mortality, clinical trial failures and post-approval withdrawal. This suggests an unmet need for improved in vitro models for DILI risk prediction that can account for diverse host genetics and other clinical factors. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system. MethodsHLOs were derived from 3 separate iPSC lines and benchmarked on two platforms for their ability to model in vitro liver function and identify hepatotoxic compounds using biochemical assays for albumin, ALT, and AST, microscopy-based morphological profiling, and single-cell transcriptomics: 1) HLOs dispersed in 384-well formatted plates and exposed to a library of compounds. 2) HLOs adapted to a liver-on-chip system. ResultsO_LIDispersed HLOs derived from the 3 iPSC lines had similar DILI predictive capacity to intact HLOs in a high-throughput screening format allowing for measurable IC50 values of compound cytotoxicity. Distinct morphological differences were observed in cells treated with drugs exerting differing mechanisms of toxicity. C_LIO_LIOn-chip HLOs significantly increased albumin production, CYP450 expression, and ALT/AST release when treated with known DILI drugs compared to dispersed HLOs and primary human hepatocytes. On-chip HLOs were able to predict the synergistic hepatotoxicity of tenofovir-inarigivir and showed steatosis and mitochondrial perturbation via phenotypic and transcriptomic analysis with exposure to FIAU and acetaminophen, respectively. C_LI ConclusionsThe high throughput and liver-on-chip system exhibit enhanced in vivo-like function and demonstrate the potential utility of these platforms for hepatotoxicity risk assessment. Tenofovir-inarigivr associated hepatotoxicity was observed and correlates with the clinical manifestation of DILI observed in patients. LAY SUMMARYIdiosyncratic (spontaneous, patient-specific) drug-induced liver injury (DILI) is difficult to study due to the lack of liver models that function as human liver tissue and are adaptable for large-scale drug screening. Human liver organoids grown from patient stem cells respond to known DILI-causing drugs in both a high-throughput and on a physiological "chip" culture system. These platforms show promise in their use as predictive model for novel drugs before entering clinical trials.

pharmacology and toxicology↗

bioRxiv2021

In vitro models of the human esophagus reveal ancestrally diverse response to injury

European Americans (EA) are more susceptible to esophageal tissue damage and inflammation when exposed to gastric acid and bile acid reflux and have a higher incidence of esophageal adenocarcinoma when compared to African Americans (AA). Population studies have implicated specific genes for these differences; however, the underlying cause for these differences is not well understood. We describe a robust long-term culture system to grow primary human esophagus in vitro, use single cell RNA sequencing to compare primary human biopsies to their in vitro counterparts, identify known and new molecular markers of basal cell types, and demonstrate that in vivo cellular heterogeneity is maintained in vitro. We further developed an ancestrally diverse biobank and a high-content, image based, screening assay to interrogate bile-acid injury response. These results demonstrated that AA esophageal cells responded significantly differently than EA-derived cells, mirroring clinical findings, having important implications for addressing disparities in early drug development pipelines.

cell biology↗

bioRxiv2020

Morphological Cell Profiling of SARS-CoV-2 Infection Identifies Drug Repurposing Candidates for COVID-19

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10-15 years from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 FDA-approved compounds and clinical candidates, we identified 17 dose-responsive compounds with in vitro antiviral efficacy in human liver Huh7 cells and confirmed antiviral efficacy in human colon carcinoma Caco-2, human prostate adenocarcinoma LNCaP, and in a physiologic relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein classically found in secretory fluids, including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19. IMPORTANCESince its emergence in China in December 2019, SARS-CoV-2 has caused a global pandemic. Repurposing of FDA-approved drugs is a promising strategy for identifying rapidly deployable treatments for COVID-19. Herein, we developed a pipeline for quantitative high-throughput image-based screening of SARS-CoV-2 infection in human cells that led to the identification of several FDA-approved drugs and clinical candidates with in vitro antiviral activity.

cell biology↗