Systematic approaches to dissect biological processes in stem cells by image-based screening

High-throughput RNAi or small molecule screens have proven to be powerful methodologies for the systematic dissection of cellular processes. In model organisms and cell lines, large-scale screens have identified key components of many cellular pathways and helped to identify novel targets in disease-relevant pathways. Image-based high-content screening has become an increasingly important tool in high-throughput screening, enabling changes in phenotype characteristics, such as cell morphology and cell differentiation, to be monitored. In this review, we discuss the use of image-based screening approaches to explore the behavior of adult, embryonic, and induced pluripotent stem cells. First, we review how current pluripotency and differentiation assays can be adapted to high-throughput formats. We then describe general aspects of image-based screening of cells and present an outlook on challenges for screening stem cells.     

A consolidated approach to analyzing data from high-throughput protein microarrays with an application to immune response profiling in humans.

Motivation: DNA microarrays are a well-known and established technology in biological and pharmaceutical research providing a wealth of information essential for understanding biological processes and aiding drug development. Protein microarrays are quickly emerging as a follow-up technology, which will also begin to experience rapid growth as the challenges in protein to spot methodologies are overcome. Like DNA microarrays, their protein counterparts produce large amounts of data that must be suitably analyzed in order to yield meaningful information that should eventually lead to novel drug targets and biomarkers. Although the statistical management of DNA microarray data has been well described, there is no available report that offers a successful consolidated approach to the analysis of high-throughput protein microarray data. We describe the novel application of a statistical methodology to analyze the data from an immune response profiling assay using human protein microarray with over 5000 proteins on each chip.     

The development of a high-content screening binding assay for the smoothened receptor

In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-μM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.     

Configuration of a high-content imaging platform for hit identification and pharmacological assessment of JMJD3 demethylase enzyme inhibitors

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.     

Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z′ factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.     

High-throughput screening of novel peptide inhibitors of an integrin receptor from the hexapeptide library by using a protein microarray chip

Protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, ProteoChip, in new drug discovery. Integrin alpha(v)beta(3) microarray immobilized on the ProteoChip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin alpha(v)beta(3)-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner and was inhibited not only by the synthetic RGD peptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and high-throughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the ProteoChip is a promising tool for high-throughput screening of lead molecules in new drug development.     

Development and validation of sandwich ELISA microarrays with minimal assay interference

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays are emerging as a strong candidate platform for multiplex biomarker analysis because of the ELISA's ability to quantitatively measure rare proteins in complex biological fluids. Advantages of this platform are high-throughput potential, assay sensitivity and stringency, and the similarity to the standard ELISA test, which facilitates assay transfer from a research setting to a clinical laboratory. However, a major concern with the multiplexing of ELISAs is maintaining high assay specificity. In this study, we systematically determine the amount of assay interference and noise contributed by individual components of a multiplexed 24-assay system. We find that nonspecific reagent cross-reactivity problems are relatively rare. We did identify the presence of contaminant antigens in a "purified antigen". We tested the validated ELISA microarray chip using paired serum samples that had been collected from four women at a 6-month interval. This analysis demonstrated that protein levels typically vary much more between individuals than within an individual over time, a result which suggests that longitudinal studies may be useful in controlling for biomarker variability across a population. Overall, this research demonstrates the importance of a stringent screening protocol and the value of optimizing the antibody and antigen concentrations when designing chips for ELISA microarrays.     

Functional assays for screening GPCR targets

G-protein-coupled receptors (GPCRs) are valuable molecular targets for drug discovery. An important aspect of the early drug discovery process is the design and implementation of high-throughput GPCR functional assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates. Several functional assay kits based on fluorescence and/or chemiluminescence detection are commercially available for convenient screen development, each having advantages and disadvantages. In addition, new GPCR biosensors and high-content imaging technologies have recently been developed that hold promise for the development of functional GPCR screens in living cells.     

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the K_on and K_off values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.     

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the K_on and K_off values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.     

Multiplexed assays by high-content imaging for assessment of GPCR activity

G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor imaging assay is based on redistribution of cytosolic beta-arrestin to an activated GPCR and has become widely used in high-content screening. However, assessing Transfluor alone has limitations: relying on a single mechanistic step of beta-arrestin redistribution during GPCR activation, providing no information on the stimulated GPCR's intracellular fate, and using only a single fluorescent color (green fluorescent protein). Taking full advantage of high-content imaging to screen approximately 2000 compounds, the authors multiplexed the Transfluor assay with an immunofluorescence-based quantification of GPCR internalization. This approach identified and classified 377 compounds interfering with agonist-induced activation of the Transfluor assay, receptor internalization, or both. In addition, a subset of compounds was analyzed for their performance across imaging, cell-based calcium release (fluorometric imaging plate reader [FLIPR]), and biochemical receptor binding assays (scintillation proximity assay). This indicated that the imaging assays have even better predictive power for direct inhibition of receptor binding than the FLIPR assay. In conclusion, compounds inducing unique responses can suggest novel mechanisms of action and be used as tools to study GPCR activation and internalization.     

Validation of affinity reagents using antigen microarrays

There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.     

Integrative computational biology for cancer research

Over the past two decades, high-throughput (HTP) technologies such as microarrays and mass spectrometry have fundamentally changed clinical cancer research. They have revealed novel molecular markers of cancer subtypes, metastasis, and drug sensitivity and resistance. Some have been translated into the clinic as tools for early disease diagnosis, prognosis, and individualized treatment and response monitoring. Despite these successes, many challenges remain: HTP platforms are often noisy and suffer from false positives and false negatives; optimal analysis and successful validation require complex workflows; and great volumes of data are accumulating at a rapid pace. Here we discuss these challenges, and show how integrative computational biology can help diminish them by creating new software tools, analytical methods, and data standards.     

Flow cytometry for high-throughput, high-content screening

Flow cytometry is a mature platform for quantitative multi-parameter measurement of cell fluorescence. Recent innovations allow up to 30-fold faster serial processing of bulk cell samples. Homogeneous discrimination of free and cell-bound fluorescent probe eliminates wash steps to streamline sample processing. Compound screening throughput may be further enhanced by multiplexing of assays on color-coded bead or cell suspension arrays and by integrating computational techniques to create smaller, focused compound libraries. Novel bead-based assay systems allow studies of real-time interactions between solubilized receptors, ligands and molecular signaling components that recapitulate and extend measurements in intact cells. These new developments, and its broad usage, position flow cytometry as an attractive analysis platform for high-throughput, high-content biological testing and drug discovery.     

Multiparameter measurement of caspase 3 activation and apoptotic cell death in NT2 neuronal precursor cells using high-content analysis

Caspase activation is a component of a number of neurodegenerative disorders, including stroke. In this study, the authors describe a multiplexed assay for caspase 3 activation, nuclear condensation, and cell viability in a neuronal precursor cell line Ntera-2, injured with staurosporine and etoposide. Using a high-content screening approach, cells were identified by staining with the nuclear stain Hoechst 33342; cell viability was measured by staining cells with YoPro-1, which is taken up by damaged cells but excluded from healthy cells; and caspase 3/7 activation was detected using the cell-permeable probe PhiPhi-Lux, which becomes fluorescent when cleaved by active caspase 3 or 7. These 3 dyes were detected simultaneously using a 4-band pass filter set on a Cellomics Array scan. The authors used peptide-fmk inhibitors selective for a variety of caspases, demonstrating that the injury is mediated primarily through caspase 3 or 7, although other caspases or related proteases may play a minor role. The general caspase inhibitor zVAD-fmkwas able to block cell death and caspase activation with the highest potency. The caspase 3 selective inhibitor DEVD-fmkwas almost as potent as zVAD-fmk; other peptide caspase inhibitors displayed only modest inhibition of cell death. This assay was also used as a high-content screening tool for the evaluation of novel caspase 3 inhibitors for the potential treatment of degenerative disorders.     

Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns

Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation.     

High-throughput proteomics using antibody microarrays

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.     

Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening

Hybridoma methods for monoclonal antibody (mAb) cloning are a mainstay of biomedical research, but they are hindered by the need to maintain hybridomas in oligoclonal pools during antibody screening. Here, we describe a system in which hybridomas specifically capture and display the mAbs they secrete: On-Cell mAb Screening (OCMS?). In OCMS?, mAbs displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS? demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG “cap”, as a universal assay for anti-viral mAbs. We produced and characterized OCMS?-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. Lastly, we used OCMS? to assess expression and antigen binding of a recombinant mAb produced in 293T cells. As a novel method to physically associate mAbs with the hybridomas that secrete them, OCMS? overcomes a central challenge to hybridoma mAb screening and offers new paradigms for mAb discovery and production.