In general, monolayer neuronal cultures are derived from dividing neuronal precursor cells that can be dissociated from each other, expanded under growth factor stimulation, and frozen, providing a useful source for later analyses. The advantages of these types of cultures are that a large number of neuronal progenitor cells can be obtained and differentiated into mature neurons, and analyses of cellular morphology (such as neuronal branching and spine quantification) are easier to perform, owing to the 2D nature of these in vitro systems. One of the most commonly used monolayer protocols in the ES and hiPSC fields is the dual SMAD inhibition method 36; the original version of this resulted in conversion of ES and hiPSCs to neuronal cells with an efficiency >80%, and an adaptation of this protocol has successfully been used to generate dopaminergic neurons to study Parkinson disease 37,38 and to generate cortical interneurons 39. An alternative protocol, that combined dual SMAD inhibition with retinoid signaling led to a 95% efficiency of neuronal differentiation 40 and has been used to model Alzheimer disease and schizophrenia 41–43. In
