High-throughput screening-compatible single-step protocol to differentiate embryonic stem cells in neurons.
Biotechnologies such as high-throughput screening (HTS) enable evaluation of large compound libraries for their biological activity and toxic properties. In the field of drug development, embryonic stem (ES) cells have been instrumental in HTS for testing the effect of new compounds. We report an innovative method in one step to differentiate ES cells in neurons and glial cells. The four different neuronal subtypes, gamma-aminobutyric acid (GABA)-ergic, dopaminergic, serotonergic, and motor neurons, are formed in culture. This protocol is adaptable to small wells and is highly reproducible, as indicated by the Z-factor value. Moreover, by using either leukemia inhibitory factor (LIF) or recombinant Cripto protein in our culture conditions, we provide evidence that this protocol is suitable for testing the effect of different molecules on neuronal differentiation of ES cells. Finally, thanks to the simplicity in carrying out the experiment, this method provides the possibility of following the morphological evolution of the in vitro differentiating neuronal cells by timelapse videomicroscopy. Our experimental system provides a powerful tool for testing the effect of different substances on survival and/or differentiation of neuronal and glial cells in an HTS-based approach. Furthermore, using genetically modified ES cells, it would be possible to screen for drugs that have a therapeutic effect on specific neuronal pathologies or to follow, by time-lapse videomicroscopy, their ability to in vitro differentiate.
Fico
Annalisa
A
Stem Cell Fate Lab, Istituto di Genetica e Biofisica Adriano Buzzati Traverso CNR, Via Pietro Castellino 111, 80131 Napoli, Italy.
Manganelli
Genesia
G
Simeone
Marino
M
Guido
Stefano
S
Minchiotti
Gabriella
G
Filosa
Stefania
S
D015415:Biomarkers / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005855:Germ Layers / Q000166:cytology / Q000187:drug effects; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D013997:Time Factors
D015415:Biomarkers; D053605:Leukemia Inhibitory Factor; D004815:Epidermal Growth Factor