Given the survival of neurons grown in the microdevice, we sought to determine if their subtype identity was preserved by a morphological analysis. We seeded inhibitory neurons in the center and excitatory and DA neurons in the outer chambers. At 4 weeks post plating, we fixed the cultures and stained for pan-neuronal and subtype-specific markers. Excitatory neurons expressed MAP2 and Tuj1, along with vGlut2, indicating an excitatory neuronal phenotype (Fig. 3a,d). DA neurons showed expression of MAP2 and TH, consistent with a dopaminergic phenotype (Fig. 3b). Inhibitory neurons expressed GAD6, the rate-limiting enzyme for the production of GABA (Fig. 3c). Human iNs also expressed synapsin (Fig. 3e,f), suggesting synapse formation within the device. While generally neurons of a specific subtype remained within their distinctive chamber, there was a small percentage (∼0.001%) of cells that apparently migrated through the microchannels. In spite of these few cells, these images demonstrate that iPSCs were converted into mature neurons, and that these three subtypes can be maintained in distinct compartments within the microdevice.
