micropatterned studies, substrates were either placed into one well of a 24-well plate or pre-designed live cell imaging substrates were used, coated with laminin and 150,000 to 300,000 neurons were seeded on day 25 to day 30 of differentiation. Once neurons attached to the surface, they separated into individual neuronal processes. We determined the specific size of single cell-adhesive areas and the distance from those areas to each other (for each flower-, star- and line-shaped patterning environment) to ensure that neurons evenly distributed onto the substrate. Under these optimized conditions, neurites were able to cross the cell-repellent regions to form connections with nearby neurites and cell bodies resulting in neuronal networks 20h after plating neurons onto a substrate with a flower-shaped pattern (Figure 2C, left image). At day 40 of the differentiation protocol, micropatterned neurons stained positive for the neuronal marker β-III-tubulin (Figure 2C, right image) as well as pre- and postsynaptic markers (data not shown). During early development iPSC-derived dopaminergic neurons initiate the expression of midbrain and dopaminergic markers, such as LMX1A, FOXA2 and tyrosine hydroxylase (TH). These markers are commonly used to assess the efficiency of neuronal differentiation. We compared neurons plated on micropatterned substrates with those grown
