Figure 3.

Differentiation of hiPSCs into MSNs. A, The WNT inhibitor IWP2 was added either at the beginning of differentiation or at day 5 and telencephalic identity was monitored by analyzing FOXG1 mRNA expression by qRT-PCR on day 5, 10, and 13. Only when IWP2 was added from day 0 the expression of the forebrain marker FOXG1 was strongly induced. Data were exemplarily generated with the hiPSC line WT1–2. B, When EBs were plated at day 8 of differentiation and fixed 2 d later, cells stained positive for the neural progenitor cell marker NESTIN and FOXG1. C, At day 21 of the differentiation process, neurons marked by the expression of TUBB3 mostly expressed FOXG1. D, At day 30, different hiPSC lines of ChAc patients (Mut 1 and 2) and controls (WT 1–1 and 1–2) showed comparable expressions of the genes FOXG1, GAD67, and MAP2 as analyzed by qRT-PCR and normalized to the average expression level in undifferentiated hiPSCs. MSNs were replated as single cells at ∼day 30. Two days later, most of the TUBB3⁺ neurons (E) stained also positive for FOXG1 and GABA and ∼40% of patient and control MSNs expressed DARPP32 (F). The stainings were performed in triplicate (FOXG1 and DARPP32) or duplicate (GABA). Data are shown as means ± SEM. For D–F, the lines WT1–1, WT1–2, Mut1, and Mut2 were used. G, Mature MSNs at ∼day 30 or older expressed TUBB3, DARPP32, MAP2, GAD67, and GABA, as shown by representative immunocytochemical staining. Scale bars, 100 μm.