To further determine whether human astrocyte-derived apoE4 also displays detrimental effects in hiPSC-derived neurons, we differentiated hiPSC with different apoE genotypes into mature astrocytes (Supplementary Fig. 12b,d,e) and collected the conditioned media (ACM) with secreted apoE3 or apoE4. The apoE−/− hiPSC-derived neurons in culture were treated for 1 week with the ACM containing 0.35 nM (12 ng/ml) or 1.47 nM (50 ng/ml) of apoE. These concentrations of apoE were similar (0.35 nM) to or 3–4-fold higher (1.47 nM) than those in brain interstitial fluid (ISF) of mice34, which largely reflect the pool of astrocyte-secreted apoE in brains. Clearly, apoE4-ACM treatment at both concentrations did not significantly affect the levels of p-tau (AT8 and PHF1), GAD67 (GABAergic neuron marker), and Aβ40 and Aβ42 production/secretion in apoE−/− hiPSC-derived neuron culture (Supplementary Fig. 13). Thus, human astrocyte-derived apoE4 does not confer detrimental effects in human neurons (at least for tau phosphorylation, GABAergic neuron degeneration, and Aβ production), supporting the conclusion that the gain of toxic effects of apoE4 is neuron specific.
