A Real-time Impedance-Based Screening Assay for Drug-Induced Vascular Leakage

Vascular leakage is a serious side effect of therapies based on monoclonal antibodies or cytokines which may lead to life-threatening situations. With the steady increase of new drug development programs for large molecules, there is an urgent need for reliable tools to assess this potential liability of new medicines in a rapid and cost-effective manner. Using human umbilical vein endothelial cells (HUVECs) as a model for endothelium, we established an impedance-based assay measuring the integrity of the endothelial cell monolayer in real time. We could demonstrate that the HUVEC monolayer in our system was a relevant model as cells expressed major junctional proteins known to be responsible for maintaining tightness as well as receptors targeted by molecules known to induce vascular leakage in vivo. We assessed the time-dependent loss of barrier function using impedance and confirmed that signals obtained corresponded well to those from standard transwell assays. We assayed a series of reference molecules which led to the expected change of barrier integrity. A nonspecific cytotoxic effect could be excluded by using human fibroblasts as a nonresponder cell line. Finally, we could show reversibility of vascular permeability induced by histamine, IL-1β, or TNF-α by coincubation with established antagonists, further demonstrating relevance of this new model. Taken together, our results suggest that impedance in combination with HUVECs as a specific model can be applied to assess clinically relevant vascular leakage on an in vitro leve

Current approaches and future role of high content imaging in safety sciences and drug discovery

High content imaging combines automated microscopy with image analysis approaches to simultaneously quantify multiple phenotypic and/or functional parameters in biological systems. The technology has become an important tool in the fields of safety sciences and drug discovery, because it can be used for mode-of-action identification, determination of hazard potency and the discovery of toxicity targets and biomarkers. In contrast to conventional biochemical endpoints, high content imaging provides insight into the spatial distribution and dynamics of responses in biological systems. This allows the identification of signaling pathways underlying cell defense, adaptation, toxicity and death. Therefore high content imaging is considered a promising technology to address the challenges for the Toxicity testing in the 21st century approach. Currently high content imaging technologies are frequently applied in academia for mechanistic toxicity studies and in pharmaceutical industry for the ranking and selection of lead drug compounds or to identify/confirm mechanisms underlying effects observed in vivo. A recent workshop gathered scientists working on high content imaging in academia, pharmaceutical industry and regulatory bodies with the objective to compile the state-of-the-art of the technology in the different institutions. They defined technical and methodological gaps, addressed the need for quality control, suggested control compounds and acceptance criteria, highlighted cell sources and new readouts and discussed future requirements for regulatory implementation. This review summarizes the discussion, proposed solutions and recommendations of the specialists contributing to the workshop.JRC.I.5-Systems Toxicolog

Human induced pluripotent stem cells generate light responsive retinal organoids with variable and nutrient dependent efficiency

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format which enhanced generation of retinal organoids with retinal pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. © AlphaMed Press 2018

Incorporating microglia‐like cells in human induced pluripotent stem cell‐derived retinal organoids

Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina

Human Retinal Organoids Provide a Suitable Tool for Toxicological Investigations: a Comprehensive Validation Using Drugs and Compounds Affecting the Retina

Retinal drug toxicity screening is essential for the development of safe treatment strategies for a large number of diseases. To this end, retinal organoids derived from human pluripotent stem cells (hPSCs) provide a suitable screening platform due to their similarity to the human retina and the ease of generation in large-scale formats. In this study, two hPSC cell lines were differentiated to retinal organoids, which comprised all key retinal cell types in multiple nuclear and synaptic layers. Single-cell RNA-Seq of retinal organoids indicated the maintenance of retinal ganglion cells and development of bipolar cells: both cell types segregated into several subtypes. Ketorolac, digoxin, thioridazine, sildenafil, ethanol, and methanol were selected as key compounds to screen on retinal organoids because of their well-known retinal toxicity profile described in the literature. Exposure of the hPSC-derived retinal organoids to digoxin, thioridazine, and sildenafil resulted in photoreceptor cell death, while digoxin and thioridazine additionally affected all other cell types, including Müller glia cells. All drug treatments caused activation of astrocytes, indicated by dendrites sprouting into neuroepithelium. The ability to respond to light was preserved in organoids although the number of responsive retinal ganglion cells decreased after drug exposure. These data indicate similar drug effects in organoids to those reported in in vivo models and/or in humans, thus providing the first robust experimental evidence of their suitability for toxicological studies

Organ-on-a-chip technologies that can transform ophthalmic drug discovery and disease modeling

Introduction: Disorders of the eye that lead to visual impairment are affecting millions of people worldwide. Nevertheless, for many of these disorders, there are still no effective treatment options available due to the lack of in vitro model systems that emulate the physiological in vivo structure and function of human eyes. Microphysiological organ-on-a-chip (OoC) technology represents a novel and powerful approach to overcome the limitations of conventional model systems and lead to a paradigm shift in ophthalmic research. Areas covered: This review provides an overview of the various tissues of interest in ophthalmology and summarizes existing model systems, including their applications and limitations. Additionally, novel OoC systems with applications in ophthalmology are described and the advantages of these systems compared to conventional models are highlighted. Expert opinion: The physiological relevance of the first ophthalmic OoC systems that mimic human ocular compartments, such as the cornea and retina, has been successfully demonstrated in recent years. There is a great potential for the application of these platforms for future pharmacological target identification, safety, and efficacy testing, as well as personalized medicine. Further improvements and the development of new systems are of upmost importance, especially to model complex disorders affecting several tissues

In vitro teratogenicity testing using a 3D, embryo-like gastruloid system

Pharmaceuticals intended for use in patients of childbearing potential need to be tested for teratogenicity before marketing. Several pharmaceutical companies use animal-free in vitro models which allow a more rapid selection of lead compounds and contribute to 3Rs principles ('replace, reduce and refine') by streamlining the selection of promising compounds submitted to further regulatory studies in animals. Currently available in vitro models typically rely on adherent monolayer cultures or disorganized 3D structures, both of which lack the spatiotemporal and morphological context of the developing embryo. A newly developed 3D 'gastruloid' model has the potential to achieve a more reliable prediction of teratogenicity by providing a robust recapitulation of gastrulation-like events alongside morphological coordination at relatively high-throughput. In this first proof-of-concept study, we used both mouse and human gastruloids to examine a panel of seven reference compounds, with associated in vivo data and known teratogenic risk, to quantitatively assess in vitro teratogenicity. We observed several gross morphological effects, including significantly reduced elongation or decreased size of the gastruloids, upon exposure to several of the reference compounds. We also observed aberrant gene expression using fluorescent reporters, including SOX2, BRA, and SOX17, suggestive of multi-lineage differentiation defects and disrupted axial patterning. Finally, we saw that gastruloids recapitulated some of the known in vivo species-specific susceptibilities between their mouse and human counterparts. We therefore suggest that gastruloids represent a powerful tool for teratogenicity assessment by enabling relevant physiological recapitulation of early embryonic development, demonstrating their use as a novel in vitro teratogenic model system.This work was supported by grants from Medical Research Council (MRC) (MR/R017190/1 and MR/V005367/1) to A.M.A. and N.M., a Leverhulme Trust grant (RPG-2018-356) to AMA, as well as a Gates Cambridge Scholarship awarded to V.M.. The mouse part was supported by an ERC Advanced investigator grant (AdG 834580) to A.M.A. supporting P.B.B. Data derived from human iPSC in vitro model based compound assessment has been supported by the BMBF, EFSA, and the DK-EPA (MST-667-00205) and received funding from the European Union's Horizon 2020 research and innovation program under grant agreements No. 681002 (EU-ToxRisk) and No. 825759 (ENDpoiNTs

Focus on germ-layer markers : A human stem cell-based model for in vitro teratogenicity testing

Human induced pluripotent stem cells (hiPSC) were used to develop an assay format that may deliver information on teratogenicity of drugs. A human pluripotent stem cell scorecard panel was used to monitor the expression of 96 marker genes that are indicative of the stem cell state or differentiation into the ectoderm, mesoderm and endoderm lineages. We selected a human episomal iPS cell line for the assay based on karyotype stability, initial pluripotency, differentiation capacity and overall gene expression variability. The assay is based on embryoid body formation and was developed to be simply automated. In this proof of concept study, we used eight reference compounds (valproic acid, all-trans-retinoic acid, thalidomide, methotrexate, hydroxyurea, ascorbic acid, penicillin G and ibuprofen) to test the technical performance of the assay (readout stability) in concentration-response and time-course experiments. We also found that each compound affected marker gene expression in a different way. Various forms of data analysis identified 19 out of 96 early developmental genes as potential predictive markers for teratogenicity. Machine-learning models were run to exemplify how the assay will be developed further. The preliminary results from these analyses suggest that the assay could be suitable for the pre-screening of candidate pharmaceutical compounds. The approach presented here points a way towards development of a human cell-based assay that could replace the murine EST currently used to screen for early indications of potential teratogenicity of drug candidates.publishe

Optimization of the TeraTox assay for preclinical teratogenicity assessment

Current animal-free methods to assess teratogenicity of drugs under development still deliver high numbers of false negatives. To improve the sensitivity of human teratogenicity prediction, we characterized the TeraTox test, a newly developed multi-lineage differentiation assay using 3D human induced pluripotent stem cells. TeraTox produces as primary output concentration-dependent cytotoxicity and altered gene expression induced by each test compound. These data are fed into an interpretable machine-learning model to perform prediction, which relates to the concentration-dependent human teratogenicity potential of drug candidates. We applied TeraTox to profile 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. Comparing TeraTox predictions with known human or animal toxicity, we report an accuracy of 69% (specificity: 53%, sensitivity: 79%). TeraTox performed better than two quantitative structure-activity relationship models, and had a higher sensitivity than the murine embryonic stem cell test (accuracy: 58%, specificity: 76%, sensitivity: 46%) run in the same laboratory. The overall prediction accuracy could be further improved by combining TeraTox and mEST results. Furthermore, patterns of altered gene expression revealed by TeraTox may help grouping toxicologically similar compounds and possibly deducing common modes of action. The TeraTox assay and the dataset described here therefore represent a new tool and a valuable resource for drug teratogenicity assessment.publishe