Three-dimensional (3D) organotypic models that capture native-like physiological features of tissues are being pursued as clinically predictive assays for therapeutics development. A range of these models are being developed to mimic brain morphology, physiology, and pathology of neurological diseases. Biofabrication of 3D gel-based cellular systems is emerging as a versatile technology to produce spatially and cell-type tailored, physiologically complex and native-like tissue models. Here we produce 3D fibrin gel-based functional neural co-culture models with human-iPSC differentiated dopaminergic or glutamatergic neurons and astrocytes. We further introduce genetically encoded fluorescence biosensors and optogenetics activation for real time functional measurements of intracellular calcium and levels of dopamine and glutamate neurotransmitters, in a high-throughput compatible plate format. We use pharmacological perturbations to demonstrate that the drug responses of 3D gel-based neural models are like those expected from in-vivo data, and in some cases, in contrast to those observed in the equivalent 2D neural models.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.

Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signalling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumours. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small-molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumour formation and slows growth of vemurafenib-resistant tumours. Our results identify an intronic mutation as the molecular basis for a RNA splicing-mediated RAF inhibitor resistance mechanism and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited monogenic disorders, characterized by a progressive decline in kidney function due in part to the formation of fluid-filled cysts. While there is one FDA-approved therapy, it is associated with potential adverse effects, and all other clinical interventions are largely supportive. Insights into the cellular pathways underlying ADPKD have revealed striking similarities to cancer. Moreover, several drugs originally developed for cancer have shown to ameliorate cyst formation and disease progression in animal models of ADPKD. These observations prompted us to develop a high-throughput screening platform of cancer drugs in a quest to repurpose them for ADPKD. We screened ~8,000 compounds, including compounds with oncological annotations, as well as FDA-approved drugs, and identified 155 that reduced the viability of Pkd1-null mouse kidney cells with minimal effects on wild-type cells. We found that 109 of these compounds also reduced in vitro cyst growth of Pkd1-null cells cultured in a 3D matrix. Moreover, the result of the cyst assay identified therapeutically relevant compounds, including agents that interfere with tubulin dynamics and reduced cyst growth without affecting cell viability. Because it is known that several ADPKD therapies with promising outcomes in animal models failed to be translated to human disease, our platform also incorporated the evaluation of compounds in a panel of primary ADPKD and normal human kidney (NHK) epithelial cells. Although we observed differences in compound response amongst ADPKD and NHK cell preparation, we identified 18 compounds that preferentially affected the viability of most ADPKD cells with minimal effects on NHK cells. Our study identifies attractive candidates for future efficacy studies in advanced pre-clinical models of ADPKD.

Use of hepatocytes derived from induced pluripotent stem cells (i-Heps) is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA) of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI) for comparing the hepatic properties of cells against a physiological gold standard. The HLI was then applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Laminin 411, the top hit, was validated in two additional induced pluripotent stem cell (iPSC) lines, primary tissue, and an in vitro model of α1-antitrypsin deficiency. Cumulatively, these data provide a reference method to control and screen for i-Hep differentiation, identify Laminin 411 as a key niche protein, and underscore the importance of combining substrates, soluble factors, and HCA when developing iPSC applications.

Determining a molecule's mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a protein's unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered to withstand unfolding at temperatures greater than 90 °C and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule.

Although acne is the most common human inflammatory skin disease, its pathogenic mechanisms remain incompletely understood. Here we show that GATA6, which is expressed in the upper pilosebaceous unit of normal human skin, is down-regulated in acne. GATA6 controls keratinocyte proliferation and differentiation to prevent hyperkeratinisation of the infundibulum, which is the primary pathological event in acne. When overexpressed in immortalised human sebocytes, GATA6 triggers a junctional zone and sebaceous differentiation program whilst limiting lipid production and cell proliferation. It modulates the immunological repertoire of sebocytes, notably by upregulating PD-L1 and IL10. GATA6 expression contributes to the therapeutic effect of retinoic acid, the main treatment for acne. In a human sebaceous organoid model GATA6-mediated down-regulation of the infundibular differentiation program is mediated by induction of TGFβ signalling. We conclude that GATA6 is involved in regulation of the upper pilosebaceous unit and may be an actionable target in the treatment of acne.

Human induced pluripotent stem cells (hiPSCs) and organoids are important for modeling human development and disease in vitro. In this study, we describe a protocol to differentiate hiPSC toward pancreatic progenitor (PP) organoids and beta-like cells. We detail the expansion and seeding of hiPSC, PP differentiation, organoid expansion, and the differentiation of PP into beta cells. Upon differentiation, organoids contained beta, delta, and alpha cells. For complete details on the use and execution of this protocol, please refer to Cujba et al. (2022).

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells.

Bardet-Biedl syndrome (BBS) is a ciliopathy with pleiotropic effects on multiple tissues, including the kidney. Here we have compared renal differentiation of iPS cells from healthy and BBS donors. High content image analysis of WT1-expressing kidney progenitors showed that cell proliferation, differentiation and cell shape were similar in healthy, BBS1, BBS2, and BBS10 mutant lines. We then examined three patient lines with BBS10 mutations in a 3D kidney organoid system. The line with the most deleterious mutation, with low BBS10 expression, expressed kidney marker genes but failed to generate 3D organoids. The other two patient lines expressed near normal levels of BBS10 mRNA and generated multiple kidney lineages within organoids when examined at day 20 of organoid differentiation. However, on prolonged culture (day 27) the proximal tubule compartment degenerated. Introducing wild type BBS10 into the most severely affected patient line restored organoid formation, whereas CRISPR-mediated generation of a truncating BBS10 mutation in a healthy line resulted in failure to generate organoids. Our findings provide a basis for further mechanistic studies of the role of BBS10 in the kidney.

The HNF1αp291fsinsC truncation is the most common mutation associated with maturity-onset diabetes of the young 3 (MODY3). Although shown to impair HNF1α signaling, the mechanism by which HNF1αp291fsinsC causes MODY3 is not fully understood. Here we use MODY3 patient and CRISPR/Cas9-engineered human induced pluripotent stem cells (hiPSCs) grown as 3D organoids to investigate how HNF1αp291fsinsC affects hiPSC differentiation during pancreatic development. HNF1αp291fsinsC hiPSCs shows reduced pancreatic progenitor and β cell differentiation. Mechanistically, HNF1αp291fsinsC interacts with HNF1β and inhibits its function, and disrupting this interaction partially rescues HNF1β-dependent transcription. HNF1β overexpression in the HNF1αp291fsinsC patient organoid line increases PDX1+ progenitors, while HNF1β overexpression in the HNF1αp291fsinsC patient iPSC line partially rescues β cell differentiation. Our study highlights the capability of pancreas progenitor-derived organoids to model disease in vitro. Additionally, it uncovers an HNF1β-mediated mechanism linked to HNF1α truncation that affects progenitor differentiation and could explain the clinical heterogeneity observed in MODY3 patients.

Joint analysis of single-cell genomics data from diseased tissues and a healthy reference can reveal altered cell states. We investigate whether integrated collections of data from healthy individuals (cell atlases) are suitable references for disease-state identification and whether matched control samples are needed to minimize false discoveries. We demonstrate that using a reference atlas for latent space learning followed by differential analysis against matched controls leads to improved identification of disease-associated cells, especially with multiple perturbed cell types. Additionally, when an atlas is available, reducing control sample numbers does not increase false discovery rates. Jointly analyzing data from a COVID-19 cohort and a blood cell atlas, we improve detection of infection-related cell states linked to distinct clinical severities. Similarly, we studied disease states in pulmonary fibrosis using a healthy lung atlas, characterizing two distinct aberrant basal states. Our analysis provides guidelines for designing disease cohort studies and optimizing cell atlas use.