GRAIL1 stabilizes misfolded mutant p53 through a ubiquitin ligase-independent, chaperone regulatory function.

Frequent (>70%) TP53 mutations often promote its protein stabilization, driving esophageal adenocarcinoma (EAC) development linked to poor survival and therapy resistance. We previously reported that during Barrett's (BE) progression to EAC, an isoform switch occurs in the E3 ubiquitin ligase RNF128 (aka GRAIL - gene related to anergy in lymphocytes), enriching isoform 1 (hereby GRAIL1) and, stabilizing the mutant p53 protein. Consequently, GRAIL1 knockdown degrades mutant p53. But how GRAIL1 stabilizes the mutant p53 protein remains unclear. In search for a mechanism, here we performed biochemical and cell biology studies to identify that GRAIL has a binding domain (315-PMCKCDILKA-325) for Hsp40/DNAJ. This interaction can influence DNAJ chaperone activity to modulate misfolded mutant p53 stability. As predicted, either the overexpression of a GRAIL fragment (Frag-J) encompassing the DNAJ binding domain, or a cell permeable peptide (Pep-J) encoding the above 10 amino acids, can bind and inhibit DNAJ-Hsp70 co-chaperone activity thus degrading misfolded mutant p53. Consequently, either Frag-J or Pep-J can reduce the survival of mutant p53 containing dysplastic BE and EAC cells and inhibit growth of patient-derived dysplastic BE organoids (PDOs) in 3D cultures. The misfolded mutant p53 targeting and growth inhibitory effects of Pep-J is comparable to simvastatin, a cholesterol lowering drug, that can degrade misfolded mutant p53 also via inhibiting DNAJA1, although by a distinct mechanism. Implications: We identified a novel ubiquitin ligase independent, chaperone regulating domain in GRAIL and further synthesized a first-in-class novel misfolded mutant p53 degrading peptide having future translational potential.

Endogenous FGFs drive ERK-dependent cell fate patterning in 2D human gastruloids.

The role of FGF is the least understood of the morphogens driving mammalian gastrulation. Here we investigated the function of FGF in a stem cell model for human gastrulation known as a 2D gastruloid. We found a ring of FGF-dependent ERK activity that closely follows the emergence of primitive streak (PS)-like cells but expands further inward. We showed that this ERK activity pattern is required for PS-like differentiation and that loss of PS-like cells upon FGF receptor inhibition can be rescued by directly activating ERK. We further demonstrated that the ERK-ring depends on localized activation of basally localized FGF receptors (FGFR) by endogenous FGF gradients. We confirm and extend previous studies in analyzing expression of FGF pathway components, showing the main receptor to be FGFR1 and the key ligands FGF2/4/17, similar to the human and monkey embryo but different from the mouse. In situ hybridization and scRNA-seq revealed that FGF4 and FGF17 expression colocalize with the PS marker TBXT but only FGF17 is maintained in nascent mesoderm and endoderm. FGF4 and FGF17 reduction both reduced ERK activity and differentiation to PS-like cells and their derivatives, indicating overlapping function. Thus, we have identified a previously unknown role for FGF-dependent ERK signaling in 2D gastruloids and possibly the human embryo, driven by a mechanism where FGF4 and FGF17 signal through basally localized FGFR1 to induce PS-like cells.

Differential Bioactivation Profiles of Different GS-441524 Prodrugs in Cell and Mouse Models: ProTide Prodrugs with High Cell Permeability and Susceptibility to Cathepsin A Are More Efficient in Delivering Antiviral Active Metabolites to the Lung.

We assessed factors that determine the tissue-specific bioactivation of ProTide prodrugs by comparing the disposition and activation of remdesivir (RDV), its methylpropyl and isopropyl ester analogues (MeRDV and IsoRDV, respectively), the oral prodrug GS-621763, and the parent nucleotide GS-441524 (Nuc). RDV and MeRDV yielded more active metabolite remdesivir-triphosphate (RDV-TP) than IsoRDV, GS-621763, and Nuc in human lung cell models due to superior cell permeability and higher susceptivity to cathepsin A. Intravenous administration to mice showed that RDV and MeRDV delivered significantly more RDV-TP to the lung than other compounds. Nevertheless, all four ester prodrugs exhibited very low oral bioavailability (<2%), with Nuc being the predominant metabolite in blood. In conclusion, ProTides prodrugs, such as RDV and MeRDV, are more efficient in delivering active metabolites to the lung than Nuc, driven by high cell permeability and susceptivity to cathepsin A. Optimizing ProTides' ester structures is an effective strategy for enhancing prodrug activation in the lung.

Nascent matrix deposition supports alveolar organoid formation from aggregates in synthetic hydrogels.

Human induced pluripotent stem cell (iPSC) derived alveolar organoids have emerged as a system to model the alveolar epithelium in homeostasis and disease. However, alveolar organoids are typically grown in Matrigel, a mouse-sarcoma derived basement membrane matrix that offers poor control over matrix properties, prompting the development of synthetic hydrogels as a Matrigel alternative. Here, we develop a two-step culture method that involves pre-aggregation of organoids in hydrogel-based microwells followed by embedding in a synthetic hydrogel that supports alveolar organoid growth, while also offering considerable control over organoid and hydrogel properties. We find that the aggregated organoids secrete their own nascent extracellular matrix (ECM) both in the microwells and upon embedding in the synthetic hydrogels. Thus, the synthetic gels described here allow us to de-couple exogenous and nascent ECM in order to interrogate the role of ECM in organoid formation.

Near-Infrared In Vivo Imaging of Claudin-1 Expression by Orthotopically Implanted Patient-Derived Colonic Adenoma Organoids.

BACKGROUND: Claudin-1 becomes overexpressed during the transformation of normal colonic mucosa to colorectal cancer (CRC). METHODS: Patient-derived organoids expressed clinically relevant target levels and genetic heterogeneity, and were established from human adenoma and normal colons. Colonoids were implanted orthotopically in the colon of immunocompromised mice. This pre-clinical model of CRC provides an intact microenvironment and representative vasculature. Colonoid growth was monitored using white light endoscopy. A peptide specific for claudin-1 was fluorescently labeled for intravenous administration. NIR fluorescence images were collected using endoscopy and endomicroscopy. RESULTS: NIR fluorescence images collected using wide-field endoscopy showed a significantly greater target-to-background (T/B) ratio for adenoma versus normal (1.89 ± 0.35 and 1.26 ± 0.06) colonoids at 1 h post-injection. These results were confirmed by optical sections collected using endomicroscopy. Optical sections were collected in vivo with sub-cellular resolution in vertical and horizontal planes. Greater claudin-1 expression by individual epithelial cells in adenomatous versus normal crypts was visualized. A human-specific cytokeratin stain ex vivo verified the presence of human tissues implanted adjacent to normal mouse colonic mucosa. CONCLUSIONS: Increased claudin-1 expression was observed from adenoma versus normal colonoids in vivo using imaging with wide field endoscopy and endomicrosopy.

A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

Development of robust antiviral assays using relevant apical-out human airway organoids.

While breakthroughs with organoids have emerged as next-generation in vitro tools, standardization for drug discovery remains a challenge. This work introduces human airway organoids with reversed biopolarity (AORBs), cultured and analyzed in a high-throughput, single-organoid-per-well format, enabling milestones towards standardization. AORBs exhibit a spatio-temporally stable apical-out morphology, facilitating high-yield direct intact-organoid virus infection. Single-cell RNA sequencing and immunohistochemistry confirm the physiologically relevant recapitulation of differentiated human airway epithelia. The cellular tropism of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains along with host response differences between Delta, Washington, and Omicron variants, as observed in transcriptomic profiles, also suggest clinical relevance. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds (remdesivir, bemnifosbuvir, and nirmatrelvir) demonstrates that AORBs efficiently predict human efficacy, comparable to gold-standard air-liquid interface cultures, but with higher throughput (∼10-fold) and fewer cells (∼100-fold). This combination of throughput and relevance allows AORBs to robustly detect false negative results in efficacy, preventing irretrievable loss of promising lead compounds. While this work leverages the SARS-CoV-2 study as a proof-of-concept application, the standardization capacity of AORB holds broader implications in line with regulatory efforts to push alternatives to animal studies.

Integrating mechanical cues with engineered platforms to explore cardiopulmonary development and disease.

Mechanical forces provide critical biological signals to cells during healthy and aberrant organ development as well as during disease processes in adults. Within the cardiopulmonary system, mechanical forces, such as shear, compressive, and tensile forces, act across various length scales, and dysregulated forces are often a leading cause of disease initiation and progression such as in bronchopulmonary dysplasia and cardiomyopathies. Engineered in vitro models have supported studies of mechanical forces in a number of tissue and disease-specific contexts, thus enabling new mechanistic insights into cardiopulmonary development and disease. This review first provides fundamental examples where mechanical forces operate at multiple length scales to ensure precise lung and heart function. Next, we survey recent engineering platforms and tools that have provided new means to probe and modulate mechanical forces across in vitro and in vivo settings. Finally, the potential for interdisciplinary collaborations to inform novel therapeutic approaches for a number of cardiopulmonary diseases are discussed.

Type I interferon signaling induces a delayed antiproliferative response in respiratory epithelial cells during SARS-CoV-2 infection.

ABSTRACT: Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain unclear. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top four genes identified in our screen encode components of the same type I interferon (IFN-I) signaling complex­IFNAR1, IFNAR2, JAK1, and TYK2. The fifth gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response in both Calu-3 cells and iPSC-derived type 2 alveolar epithelial cells. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.

Integrative genome-scale analyses reveal post-transcriptional signatures of early human small intestinal development in a directed differentiation organoid model.

BACKGROUND: MicroRNAs (miRNAs) are important post-transcriptional gene regulators controlling cellular lineage specification and differentiation during embryonic development, including the gastrointestinal system. However, miRNA-mediated regulatory mechanisms involved in early embryonic development of human small intestine (SI) remains underexplored. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage. RESULTS: We leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Small RNA-seq profiling revealed temporal dynamics of miRNA signatures across different developmental events of the model, including definitive endoderm formation, SI lineage specification and SI regional patterning. Our multi-omic, integrative analyses showed further that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut master marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development. CONCLUSION: The present study uncovers a novel facet of miRNAs in regulating prenatal SI development. We leveraged multi-omic, systems biology approaches to discover candidate miRNA regulators associated with early SI developmental events in a human organoid model. In this study, we highlighted miRNA-mediated post-transcriptional regulation relevant to the event of SI lineage specification. The candidate miRNA regulators that we identified for the other stages of SI development also warrant detailed characterization in the future.

TGFB1 induces fetal reprogramming and enhances intestinal regeneration.

The gut epithelium has a remarkable ability to recover from damage. We employed a combination of high-throughput sequencing approaches, mouse genetics, and murine and human organoids and identified a role for TGFB signaling during intestinal regeneration following injury. At 2 days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. The depletion of macrophages or genetic disruption of TGFB signaling significantly impaired the regenerative response. Intestinal regeneration is characterized by the induction of a fetal-like transcriptional signature during repair. In organoid culture, TGFB1 treatment was necessary and sufficient to induce the fetal-like/regenerative state. Mesenchymal cells were also responsive to TGFB1 and enhanced the regenerative response. Mechanistically, pro-regenerative factors, YAP/TEAD and SOX9, are activated in the epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for cellular therapy.

Near-Infrared Imaging of Colonic Adenomas In Vivo Using Orthotopic Human Organoids for Early Cancer Detection.

Colorectal cancer is a leading cause of cancer-related morbidity and mortality worldwide. Premalignant lesions that are flat and subtle in morphology are often missed in conventional colonoscopies. Patient-derived adenoma colonoids with high and low cMet expression and normal colonoids were implanted orthotopically in the colon of immunocompromised mice to serve as a preclinical model system. A peptide specific for cMet was labeled with IRDye800, a near-infrared (NIR) fluorophore. This peptide was administered intravenously, and in vivo imaging was performed using a small animal fluorescence endoscope. Quantified intensities showed a peak target-to-background ratio at ~1 h after intravenous peptide injection, and the signal cleared by ~24 h. The peptide was stable in serum with a half-life of 3.6 h. Co-staining of adenoma and normal colonoids showed a high correlation between peptide and anti-cMet antibody. A human-specific cytokeratin stain verified the presence of human tissues implanted among surrounding normal mouse colonic mucosa. Peptide biodistribution was consistent with rapid renal clearance. No signs of acute toxicity were found on either animal necropsy or serum hematology and chemistries. Human colonoids provide a clinically relevant preclinical model to evaluate the specific uptake of a NIR peptide to detect premalignant colonic lesions in vivo.

Opposing roles for TGFß- 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 TGFß- and BMP-signaling, where inhibition of TGFß- 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 TGFß-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 TGFß- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.

IL-13-induced STAT3-dependent signaling networks regulate esophageal epithelial proliferation in eosinophilic esophagitis.

BACKGROUND: Basal zone hyperplasia (BZH) and dilated intercellular spaces (DISs) are thought to contribute to the clinical manifestations of eosinophilic esophagitis (EoE); however, the molecular pathways that drive BZH remain largely unexplored. OBJECTIVE: We sought to define the role of IL-13-induced transcriptional programs in esophageal epithelial proliferation in EoE. METHODS: We performed RNA sequencing, bioinformatics, Western blot, reverse transcriptase quantitative PCR, and histologic analyses on esophageal biopsies from healthy control and patients with EoE, primary esophageal cells derived from patients with EoE, and IL-13-stimulated esophageal epithelial keratinocytes grown at the air-liquid interface (EPC2-ALI). Genetic (shRNA) and pharmacologic (proteolysis-targeting chimera degrader) approaches and in vivo model of IL-13-induced esophageal epithelial remodeling (Krt5-rtTA x tetO-IL-13Tg) were used to define the role of signal transducer and activator of transcription 3 (STAT3) and STAT6 and secreted frizzled-related protein 1 (SFRP1) in esophageal epithelial proliferation. RESULTS: RNA-sequencing analysis of esophageal biopsies (healthy control vs EoE) and EPC2-ALI revealed 82 common differentially expressed genes that were enriched for putative STAT3 target genes. In vitro and in vivo analyses revealed a link between IL-13-induced STAT3 and STAT6 phosphorylation, SFRP1 mRNA expression, and esophageal epithelial proliferation. In vitro studies showed that IL-13-induced esophageal epithelial proliferation was STAT3-dependent and regulated by the STAT3 target SFRP1. SFRP1 mRNA is increased in esophageal biopsies from patients with active EoE compared with healthy controls or patients in remission and identifies an esophageal suprabasal epithelial cell subpopulation that uniquely expressed the core EoE proinflammatory transcriptome genes (CCL26, ALOX15, CAPN14, ANO1, and TNFAIP6). CONCLUSIONS: These studies identify SFRP1 as a key regulator of IL-13-induced and STAT3-dependent esophageal proliferation and BZH in EoE and link SFRP1+ esophageal epithelial cells with the proinflammatory and epithelial remodeling response in EoE.

Regionally distinct progenitor cells in the lower airway give rise to neuroendocrine and multiciliated cells in the developing human lung.

Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR. To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.

Opposing roles for TGFß- 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 TGFß- and BMP-signaling, where inhibition of TGFß- 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 TGFß-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 TGFß- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro .

Transplanted human intestinal organoids: a resource for modeling human intestinal development.

The in vitro differentiation of pluripotent stem cells into human intestinal organoids (HIOs) has served as a powerful means for creating complex three-dimensional intestinal structures. Owing to their diverse cell populations, transplantation into an animal host is supported with this system and allows the temporal formation of fully laminated structures, including crypt-villus architecture and smooth muscle layers that resemble native human intestine. Although the endpoint of HIO engraftment has been well described, here we aim to elucidate the developmental stages of HIO engraftment and establish whether it parallels fetal human intestinal development. We analyzed a time course of transplanted HIOs histologically at 2, 4, 6 and 8 weeks post-transplantation, and demonstrated that HIO maturation closely resembles key stages of fetal human intestinal development. We also utilized single-nuclear RNA sequencing to determine and track the emergence of distinct cell populations over time, and validated our transcriptomic data through in situ protein expression. These observations suggest that transplanted HIOs do indeed recapitulate early intestinal development, solidifying their value as a human intestinal model system.

A Pilot Study: Transcriptional Profiling, Functional Analysis, and Organoid Modeling of Intestinal Mucosa in Hirschsprung Disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital colonic aganglionosis. Many HSCR patients develop enterocolitis despite surgical resection. The pathophysiology of this inflammatory process is poorly understood. We compared transcriptional profiles and function of ganglionic and aganglionic tissue in HSCR patients. METHODS: RNA sequencing was performed on mucosal tissues from HSCR patients (n = 6) and controls (n = 3). Function of matched ganglionic and aganglionic regions were investigated utilizing organoids generated from these tissues. RESULTS: Transcriptional differences observed in ganglionic and aganglionic regions of HSCR patients included upregulation of genes involving inflammation, cell differentiation and proliferation as well as decreased expression of genes encoding mucins compared to controls. Organoids derived from ganglionic and aganglionic regions of HSCR patients were similar in epithelial cell differentiation, epithelial barrier formation and response to stimulation with bacterial metabolites and pro-inflammatory cytokines. CONCLUSIONS: Despite normal ganglionic structure, the section of colon adjacent to the aganglionic region in HSCR patients has perturbed gene expression which resembles the aganglionic segment. Transcriptional and functional changes in colonic epithelium are persevered in the ganglionic colon used for pull-through surgery. This may explain persistence of enterocolitis despite surgical excision of aganglionic colon and subsequent endorectal pull-through performed with ganglionic colon during correction of HSCR. LEVEL OF EVIDENCE: N/A.

A human liver organoid screening platform for DILI risk prediction.

BACKGROUND & AIMS: Drug-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. METHODS: HLOs were derived from three 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, AST, microscopy-based morphological profiling, and single-cell transcriptomics: i) HLOs dispersed in 384-well-formatted plates and exposed to a library of compounds; ii) HLOs adapted to a liver-on-chip system. RESULTS: Dispersed HLOs derived from the three iPSC lines had similar DILI predictive capacity as 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. On-chip HLOs significantly increased albumin production, CYP450 expression, and ALT/AST release when treated with known hepatoxic drugs compared to dispersed HLOs and primary human hepatocytes. On-chip HLOs were able to predict the synergistic hepatotoxicity of tenofovir-inarigivir and displayed steatosis and mitochondrial perturbation, via phenotypic and transcriptomic analysis, on exposure to fialuridine and acetaminophen, respectively. CONCLUSIONS: The high-throughput and liver-on-chip systems exhibit enhanced in vivo-like functions and demonstrate the potential utility of these platforms for DILI risk assessment. Tenofovir-inarigivr-associated hepatotoxicity was observed and correlates with the clinical manifestation of DILI observed in patients. IMPACT AND IMPLICATIONS: Idiosyncratic (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 for researchers in their use as predictive models for novel drugs before entering clinical trials and as a potential in vitro diagnostic tool. Our findings support further development of patient-derived liver organoid lines and their use in the context of DILI research.