We trans-differentiated human ES cells into human excitatory neurons using transient expression of Ngn2 (Fig. 1A, 1B, S1A; Zhang et al., 2013). In previous studies (e.g., Pak et al., 2015; Patzke et al., 2016), we co-cultured human neurons with mouse glia because glia secretes multiple factors that support survival and synaptogenesis (Ullian et al., 2001). However, here we aimed to develop an approach that would allow examining the effects of glial factors on neurons, making it necessary to culture human neurons without glia. Towards this goal, we compared human neurons cultured on glia with neurons cultured on mouse embryonic fibroblasts (MEFs), or on matrigel alone (Fig. 1B, 1C). We found that under all conditions, human neurons appeared to develop normally with no major change in dendritic arborization, but neurons plated on matrigel alone did not survive as well as neurons co-cultured with glia or MEFs (Fig. 1C). Therefore, we utilized human neurons cultured on MEFs as a standard approach.
