Immunofluorescent staining revealed significantly fewer GABA-positive neurons derived from apoE4/4-hiPSCs than from apoE3/3-hiPSCs, both in individual lines (Supplementary Fig. 2i) and as shown by mean values (Fig. 3a–c). Levels of GAD65/67 (GABAergic neuron marker) were lower in neuronal cultures derived from apoE4/4-hiPSCs than in those derived from apoE3/3-hiPSCs, both in individual lines (Supplementary Fig. 2j) and as shown by mean values (Fig. 3d,e). Immunostaining revealed no significant difference in the numbers of GABA-positive neurons at early time points (within 4 weeks) in culture between apoE4/4-hiPSCs and apoE3/3-hiPSCs (relative to apoE3/3 as 1 ± 0.11, mean ± SEM, n = 5 fields with total of 570 GABA+ neurons counted, apoE4/4 is 0.98 ± 0.14, mean ± SEM, n = 8 fields with total of 894 GABA+ neurons counted), suggesting that the detrimental effect of apoE4 on GABAergic neurons in culture is not a developmental effect. The decrease in GABAergic neuron numbers occurred during the late stage of neuronal differentiation of apoE4/4-hiPSCs, suggesting that apoE4 induces GABAergic neurodegeneration/loss. Importantly, apoE3/3-hiPSCs and apoE4/4-hiPSCs had similar efficiencies in generating Tbr1-positive glutamatergic neurons and TH-positive
