Traditionally, hiPSCs have been differentiated to excitatory neurons via the addition of growth factors and small molecules that modulate developmental signaling pathways. In this process known as “directed differentiation”, hiPSCs are first neuralized by dual SMAD inhibition [8], yielding a transient stage from which it is possible to dissociate and expand neural rosettes as neural progenitor cells (NPCs) [9–11]. These NPCs can be subsequently differentiated into neurons that attain characteristics of functional neurons within two to three months [12]. Such “directed differentiation” protocols are thought to recapitulate in vivo development, generating neurons that most resemble fetal forebrain tissue [13,14]. Unfortunately, directed differentiation generally yields heterogeneous neuronal populations that require long-term culture to reach maturity [14–16].
