Phenotypic Characterization of Toxic Compound Effects on Liver Spheroids Derived from iPSC Using Confocal Imaging and Three-Dimensional Image Analysis.

Cell models are becoming more complex to better mimic the in vivo environment and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest in exploring the use of three-dimensional (3D) spheroids for modeling developmental and tissue biology with the goal of accelerating translational research in these areas. Accordingly, the development of high-throughput quantitative assays using 3D cultures is an active area of investigation. In this study, we have developed and optimized methods for the formation of 3D liver spheroids derived from human iPS cells and used those for toxicity assessment. We used confocal imaging and 3D image analysis to characterize cellular information from a 3D matrix to enable a multi-parametric comparison of different spheroid phenotypes. The assay enables characterization of compound toxicities by spheroid size (volume) and shape, cell number and spatial distribution, nuclear characterization, number and distribution of cells expressing viability, apoptosis, mitochondrial potential, and viability marker intensities. In addition, changes in the content of live, dead, and apoptotic cells as a consequence of compound exposure were characterized. We tested 48 compounds and compared induced pluripotent stem cell (iPSC)-derived hepatocytes and HepG2 cells in both two-dimensional (2D) and 3D cultures. We observed significant differences in the pharmacological effects of compounds across the two cell types and between the different culture conditions. Our results indicate that a phenotypic assay using 3D model systems formed with human iPSC-derived hepatocytes is suitable for high-throughput screening and can be used for hepatotoxicity assessment in vitro.

High-content assays for characterizing the viability and morphology of 3D cancer spheroid cultures.

There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translation research. Development of higher throughput assays to quantify phenotypic changes in spheroids is an active area of investigation. The goal of this study was to develop higher throughput high-content imaging and analysis methods to characterize phenotypic changes in human cancer spheroids in response to compound treatment. We optimized spheroid cell culture protocols using low adhesion U-bottom 96- and 384-well plates for three common cancer cell lines and improved the workflow with a one-step staining procedure that reduces assay time and minimizes variability. We streamlined imaging acquisition by using a maximum projection algorithm that combines cellular information from multiple slices through a 3D object into a single image, enabling efficient comparison of different spheroid phenotypes. A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes. We report a number of readouts, including quantification of marker-specific cell numbers, measurement of cell viability and apoptosis, and characterization of spheroid size and shape. Assay performance was assessed using established anticancer cytostatic and cytotoxic drugs. We demonstrated concentration-response effects for different readouts and measured IC50 values, comparing 3D spheroid results to two-dimensional cell cultures. Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids. The proposed methods can increase performance and throughput of high-content assays for compound screening and evaluation of anticancer drugs with 3D cell models.

High-content high-throughput assays for characterizing the viability and morphology of human iPSC-derived neuronal cultures.

Abstract Development of quantitative high-throughput in vitro assays that enable assessment of viability and morphological changes in neuronal cells is an active area of investigation in drug discovery and environmental chemical safety assessment. High-content imaging is an emerging and efficient tool for generating multidimensional quantitative cellular readouts; in addition, human induced pluripotent stem cell (iPSC)-derived neurons are a promising in vitro model system that emulates both the functionality and behavior of mature neurons, and they are available in quantities sufficient for screening workflows. The goal of this study was to develop high-content imaging and analysis methods to assess multiple phenotypes in human iPSC-derived neuronal cells. Specifically, we optimized cell culture, staining, and imaging protocols in a 384-well assay format and improved laboratory workflow by designing a one-step procedure to reduce assay time and minimize cell disturbance. Phenotypic readouts include quantitative characterization of neurite outgrowth and branching, cell number and viability, as well as measures of adverse effects on mitochondrial integrity and membrane potential. To verify the robustness of the workflow, we tested a series of compounds that are established toxicants. We report concentration-response effects of selected test compounds on human iPSC-derived neuronal cells and illustrate how the proposed methods may be used for high-content high-throughput compound toxicity screening and safety evaluation of drugs and environmental chemicals.

Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson's disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this "stem cell pathology" could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD.

High-content assays for hepatotoxicity using induced pluripotent stem cell-derived cells.

Development of predictive in vitro assays for early toxicity evaluation is extremely important for improving the drug development process and reducing drug attrition rates during clinical development. High-content imaging-based in vitro toxicity assays are emerging as efficient tools for safety and efficacy testing to improve drug development efficiency. In this report we have used an induced pluripotent stem cell (iPSC)-derived hepatocyte cell model having a primary tissue-like phenotype, unlimited availability, and the potential to compare cells from different individuals. We examined a number of assays and phenotypic markers and developed automated screening methods for assessing multiparameter readouts of general and mechanism-specific hepatotoxicity. Endpoints assessed were cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis. We assayed compounds with known mechanisms of toxicity and also evaluated a diverse hepatotoxicity library of 240 compounds. We conclude that high-content automated screening assays using iPSC-derived hepatocytes are feasible, provide information about mechanisms of toxicity, and can facilitate the safety assessment of drugs and chemicals.

Multiparameter in vitro assessment of compound effects on cardiomyocyte physiology using iPSC cells.

A large percentage of drugs fail in clinical studies due to cardiac toxicity; thus, development of sensitive in vitro assays that can evaluate potential adverse effects on cardiomyocytes is extremely important for drug development. Human cardiomyocytes derived from stem cell sources offer more clinically relevant cell-based models than those presently available. Human-induced pluripotent stem cell-derived cardiomyocytes are especially attractive because they express ion channels and demonstrate spontaneous mechanical and electrical activity similar to adult cardiomyocytes. Here we demonstrate techniques for measuring the impact of pharmacologic compounds on the beating rate of cardiomyocytes with ImageXpress Micro and FLIPR Tetra systems. The assays employ calcium-sensitive dyes to monitor changes in Ca(2+) fluxes synchronous with cell beating, which allows monitoring of the beat rate, amplitude, and other parameters. We demonstrate here that the system is able to detect concentration-dependent atypical patterns caused by hERG inhibitors and other ion channel blockers. We also show that both positive and negative chronotropic effects on cardiac rate can be observed and IC(50) values determined. This methodology is well suited for safety testing and can be used to estimate efficacy and dosing of drug candidates prior to clinical studies.

A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability.

Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

Stable, sensitive, fluorescence-based method for detecting cAMP.

cAMP is a universal secondary messenger that connects changes in the extracellular environment, as detected by cell surface receptors, to transcriptional changes in the nucleus. Since cAMP-mediated signal transduction plays a role in critical cell functions and human diseases, monitoring its activity can aid in understanding these responses and the process of drug discovery. This report examines the performance of a fluorescence-based competitive immunoassay in 384-well microplate format. Using purified cAMP as a competitor the estimated detection limit was determined to be 0.1 nM and Z'-factor was greater than 0.83, which indicates that the assay is of high quality and one of the most sensitive assays currently on the market. Of note, the results obtained were similar whether the reaction was allowed to proceed for 10 min or up to 60 min. Next, HEK 293 cells were treated with the promiscuous adenylate cyclase activator, forskolin, and the beta-adrenoceptor agonist, isoproterenol. The resultant average EC50 values were 11 microM and 123 nM, respectively, which correspond to those found in the literature. Together, these results demonstrate that this assay is afast, accurate, non-radioactive method that is ideal for high-throughput screening.