Figure 1: Functionally mature midbrain-like DA neurons present in both D398 and N398 DA cultures.
From: Increased nicotine response in iPSC-derived human neurons carrying the CHRNA5 N398 allele
Representative iPSC cultures from (A) D398 and (B) N398 groups stained positively for pluripotency markers TRA-1-60 (green) and Oct4 (red). The majority of cells in iPSC-derived DA cultures expressed both MAP2+ (green) cells and TH (red) in both D398 (C) and N398 (D) groups. (E,F) Punctate staining indicates the presence of DAT (green), likely localized to synapses and surrounding TH+ cells. (G,H) Few TH+ were labeled with a pulse of EdU (green), indicating that most cells were postmitotic. (I,J) HPLC traces showing release of DA into culture medium with 1 mM nicotine. (K–P) Identification of alternative neuronal subtypes in mDA cultures. Cultures were stained with TH (red) and either (K,L) 5HT, (M,N) GAD6, or (O,P) VGluT1 (green). (Q) qPCR analysis of nAChR subunit-encoding mRNAs a3,a4,a5,a6, b2, b4, of D398 (red) or N398 (blue), for either iPSC (lighter) or DA (darker) cultures. mRNA levels are normalized to GAPDH and human fetal VTA RNA. Cultures were also assayed by qPCR for mRNAs encoding (R) TH and the midbrain marker PITX3. For all qPCR assays, DA neurons were different from iPSC cultures (ANOVA, p<0.05) but there was no difference between N398 and D398.
