ApoE stimulates Aβ-production with an ApoE4>ApoE3>ApoE2 potency rank order in human neurons cultured in the absence of glia or serum(A) Experimental design. Human neurons (iN cells) were generated from H1 ES cells by forced expression of neurogenin-2 (Ngn2), and cultured either on mouse glia (green) which secrete copious amounts of ApoE, on murine embryonic fibroblasts (MEFs) which secrete no ApoE (blue), or on matrigel (black lines).(B) Representative images of human neurons at day 10 after induction (D10) cultured on glia, MEFs, or matrigel, and sparsely labeled by EGFP transfection.(C) Survival, soma size, and neurite length of human neurons cultured on glia, MEFs, or matrigel.(D) Screening 24 secreted proteins that are abundantly produced by cultured mouse glia reveals three factors (ApoE, IGF2, and IGFBP2) that induce Aβ40 and Aβ42 secretion from human neurons cultured on MEFs. Various factors were produced as human proteins in HEK293 cells (names reflect gene symbols), and added to human neurons on MEFs at D10. Media from treated neurons were analyzed by ELISA at D12.(E) ApoE2, ApoE3, and ApoE4 (all at 10 µg/ml; from D10-12) differentially stimulate Aβ40 and Aβ42 secretion by human neurons cultured on MEFs with an ApoE4>ApoE3>ApoE2 potency rank order, thereby partially rescuing the decrease in Aβ40 and Aβ42 secretion when neurons are co-cultured with MEFs instead of glia. Summary graphs show total concentrations of Aβ (left), Aβ40 (left center), and Aβ42 (right middle) in the medium, and of the Aβ42/Aβ40 ratio (right) measured by ELISA at D12. For cellular Aβ, Aβ40, and Aβ42 levels and for similar results for neurons generated from iPS cells, see Fig. S1.Data are means ± SEM (n≥3 independent experiments); statistical significance (*, p<0.05, **, p<0.01; ***, p<0.001) was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. For additional data and controls, see Fig. S1
