Several methods have been described to obtain neurons of spe

Several methods have been described to obtain neurons of specific subtypes through differentiation of hPSCs, either via formation of three-dimensional (3D) embryoid bodies (EBs) or using monolayers as starting material (Amoroso et al., 2013; Boissart et al., 2013; Boulting et al., 2011; Eiraku and Sasai, 2012; Eiraku et al., 2008; Espuny-Camacho et al., 2013; Hu and Zhang, 2009; Kim et al., 2014; Li et al., 2009; Qu et al., 2014; Shi et al., 2012; Zeng et al., 2010). An alternative approach is transcriptional programming, whereby the forced overexpression of a cocktail of transcription factors instructs PSCs, fibroblasts, or other cell populations to adopt a specific neuronal fate (Hester et al., 2011; Vierbuchen et al., 2010). These methods have provided important insights into human neurogenesis and the pathogenesis of neurodevelopmental disorders, but they have limitations. For instance, EB-based protocols generally have comparatively low efficiencies (10%–40%) and require a relatively long time in culture to generate functional motor neurons. In addition, the neurons generated often require cellular feeder layers to survive for longer times in culture (Hu and Zhang, 2009; Boulting et al., 2011; Amoroso et al., 2013). Moreover, EB methods typically result in the formation of spheres of cells varying in size and shape, leading to differences in the kinetics and efficiency of differentiation within individual plates and from experiment to experiment. Monolayer-based protocols for the generation of both cortical and motor neurons have also been published, with recent work describing improved efficiencies (Qu et al., 2014). However, a disadvantage of this adherent monolayer-based protocol is that the neurons need to be passaged, and successful long-term culture after replating has not been described. Another common theme in the field has been the problem of obtaining mature cells from hPSCs. It has been shown that maintaining differentiated cells in culture can be challenging, thereby precluding experiments studying aspects of cellular functions that take longer times to manifest (Bellin et al., 2012; Grskovic et al., 2011). Recently, a 3D culture system that yields Cy3 hydrazide tissue from hPSCs in the form of neural organoids has been described (Bershteyn and Kriegstein, 2013; Lancaster et al., 2013; Sasai, 2013). These organoids produce neurons organized in a manner reminiscent to what is seen in distinct anatomical structures within the mammalian CNS. At least some of the neurons in the organoids are functional, and this method has thereby offered a promising approach to study neurodevelopmental mechanisms and disorders. However, at this point, formation of neural organoids is not a process that is fully controlled. Another promising recent report based on a scaffold-free plate-based 3D method used to generate spheroids showed the possibility of yielding functional neurons with properties of deep and superficial cortical neurons (Pasca et al., 2015). However, this method may be difficult to implement for large-scale production of neurons and also generates cellular structures that are large enough to be potentially subject to necrosis in the core regions of the spheroids. Here, we describe a method for large-scale production of neurons from multiple lines of human embryonic stem cells (hESCs) and human induced PSCs. We show that our method, based on the differentiation of 3D hPSC spheres maintained in suspension in spinner flasks (hereafter referred to as spin cultures), gives a higher purity and larger absolute number of cells, and has the potential to make functional neurons that can be maintained in culture for extended periods of time. Importantly, we have applied this method to the production of both cortical neurons of multiple types and spinal cord motor neurons. For both neuronal subtypes, we were able to document not only expression of appropriate marker genes but also several characteristics of mature neurons. The obtained neurons respond robustly to depolarization, form synapses as determined by punctate staining with antibodies against established synaptic proteins, and exhibit spontaneous neural activity assessed by electrophysiology and fluctuations in intracellular Ca2+ levels. Neurons generated from spheres also exhibited increased survival after dissociation compared with EB-derived neurons, and could be kept in culture without glial feeder layers for up to 5 months without compromising their functional properties. Taken together, these data illustrate that this protocol robustly generates large numbers of functional neurons that can reach a high degree of maturation, and should offer an improved cellular source of human neurons for disease modeling and drug screening.