To examine the purity of iN cultures, we tested for the presence of inhibitory GABAergic neurons, dopaminergic neurons and astrocytes via expression-based and immunohistochemical approaches. Three-week-old mNgn2-induced neurons showed reduced GAD67 (and comparably low levels of vGAT expression) relative to six-week-old hiPSC forebrain neurons (1.5-fold, p < 0.005), lower TH levels (6.3-fold, p < 0.005), and comparably low TPH1 (Fig. 4D). mNgn2-induced neurons also showed reduced levels of GFAP and S100β (13.6-fold, p < 0.001 and 3.2-fold, p < 0.001, respectively) (Fig. 4D). By immunohistochemistry, we failed to detect GAD65/67 or TH-positive neurons, or GFAP-positive astrocytes, in mNgn2-induced cultures (Fig. 4E). Taken together, these data suggest that, as predicted from the hiPSC data [19], mNgn2 overexpression primarily induces excitatory neurons.
