(A) Inhibitory neuron cultures derived from control iPSC line 8402–2 WT9 were fixed before plating onto rat astrocytes and stained for NKX2.1 (in green, using Novacastra antibody TTF‐1‐L‐CE), Calretinin (in red, using Swant antibody 7697) and nuclei (Hoechst, in blue). The arrows point to cells with neuronal morphology and co-expressing NKX2.1 and Calretinin. Scale bars, 50 microns. (B) The cells described in (A) were further differentiated in the presence of rat astrocytes and stained for GAD67 (in white, using EMD Millipore antibody AB5406) and SP8 (in red, using Sigma antibody HPA054006). In a total of 199 GAD67+, neuronal-looking cells that we analyzed, 81% (161 out of 199) was negative for SP8 staining, and the remaining 19% (38 out of 199) showed a very weak signal (one example shown in (B), highlighted by open arrow head). Scale bar, 100 microns. (C) As a positive control for SP8 detection, we examined primary cultures of rat cortical neurons (three weeks in vitro) in the same experiment under identical staining and imaging conditions. Robust SP8 staining was seen in a subpopulation of GAD67+ inhibitory neurons (arrow head). Scale bar, 100 microns. Because the mutually exclusive expression of SP8 and NKX2.1 distinguishes between the CGE- and MGE-origin of inhibitory interneurons, this set of supplementary data further support that the human iPSC-derived inhibitory neurons in this study mostly resemble a MGE origin, and that Calretinin expression may arise in MGE-lineage inhibitory neurons within the time scale of the study.
DOI: http://dx.doi.org/10.7554/eLife.13073.016