We also observed a marked reduction in the percentage of cells expressing GAD67 and Citrine between D54 and 100, likely because GAD67 and DCX are predominately expressed by postmitotic cells, and mitotic cells outnumber differentiated neurons over time. Reductions in GAD67+ cells have also been observed in vivo, and it is possible in vitro-derived cells undergo a similar process of programmed cell death (Southwell et al., 2012). It is also possible that some interneuron subtypes require excitatory input for survival and maturation, rendering monocultures suboptimal for survival and function of GABAergic neurons (Close et al., 2012; De Marco Garcia et al., 2011). These factors must be taken into account, as a clear understanding of the molecular mechanisms of subtype specification relies on the ability to produce these cells in the first place.
