RESUMEN
Stem cells hold great potential in drug discovery and development. However, challenges remain to quantitatively measure the functions of stem cells and their differentiated products. Here, we applied fluorescent imaging, quantitative real-time PCR, and label-free dynamic mass redistribution (DMR) assays to characterize the differentiation process of the ReNcell VM human neural progenitor stem cell. Immunofluorescence imaging showed that after growth factor withdrawal, the neuroprogenitor stem cell was differentiated into dopaminergic neurons, astrocytes, and oligodendrocytes, thus creating a neuronal cell system. High-performance liquid chromatography analysis showed that the differentiated cell system released dopamine upon depolarization with KCl. In conjunction with quantitative real-time PCR, DMR assays using a G-protein-coupled receptor agonist library revealed that a subset of receptors, including dopamine D(1) and D(4) receptors, underwent marked alterations in both receptor expression and signaling pathway during the differentiation process. These findings suggest that DMR assays can decode the differentiation process of stem cells at the cell system level.
Asunto(s)
Imagen Molecular/métodos , Células-Madre Neurales/metabolismo , Neurogénesis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Astrocitos/citología , Astrocitos/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Dopamina/análisis , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Oligodendroglía/citología , Oligodendroglía/metabolismo , Cloruro de Potasio/farmacología , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D4/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The ability to combine primary hit identification assays with target profiling would significantly streamline the current drug discovery process. Working towards this end, we report here the development of a microarray-based ligand binding assay that supports multiplexed analysis of G protein-coupled receptor systems in a 96-well microplate format that is compatible with the equipment and infrastructure typical of high-throughput screening laboratories. A prototype microarray was generated by pin-printing seven different receptors within the wells of a specially coated glass-bottom microplate and assaying with a cocktail of fluorescent ligands. Development of the multiplexed system included optimization of methods for depositing receptor membrane proteins and establishing a generic set of assay conditions that simultaneously satisfied the pharmacology requirements of all of the receptor systems included on the array. The multiplexed system is shown to produce valid pharmacological results as evidenced by its ability to report K(i) values for receptor-specific fluorescent ligands and rank ordered potencies for diagnostic displacing compounds comparable to values generated by conventional simplexed assays. Moreover, the results of a 40-compound mini-screen confirmed that the assay accurately identifies valid hits. The results suggest the assay may be immediately suitable for routine profiling tasks and demonstrate the potential of the format for high-throughput multiplexed drug discovery.
Asunto(s)
Evaluación Preclínica de Medicamentos , Análisis por Micromatrices/métodos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Tampones (Química) , Interpretación Estadística de Datos , Dimetilsulfóxido/química , Diseño de Fármacos , Perfilación de la Expresión Génica , Humanos , Indicadores y Reactivos , Ligandos , Unión Proteica , Proteínas/química , Receptores de Droga/química , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SolventesRESUMEN
Conventional assay methods for discovering and profiling drug-target interactions are typically developed on a target-by-target basis and hence can be cumbersome to enable and orchestrate. Herein the authors report a solid-state ligand-binding assay that operates in a multiplexed mode to report compound activity against a micorarray-configured panel of G-protein-coupled receptor (GPCR) targets. The pharmacological fidelity of the system is high, and its miniaturized "plug-and-play" format provides improved efficiency both in terms of execution time and reagent consumption. Taken together, these features make the system ideally suited to explore the structure-activity relationship of compounds across a broad region of target class space.