fig2

Figure 2. Brief protocol for directed differentiation of glutamatergic neurons from human induced pluripotent stem cell (hiPSC). Undifferentiated hiPSC are grown in E8 media (dark orange bar) to 80% confluency, whereupon they are exposed to the dual SMAD inhibitors (LDN193189 and SB431542) until day 10. Afterwards, proliferation of these neural progenitors (NPCs) are stimulated using fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). A critical point is not to exceed passage 4 to avoid a shift to glial progenitors. After day 17 NPCs are matured in a medium containing brain-derived neurotrophic factor, glial cell-derived neurotrophic factor (GDNF), dibutyryl-cAMP (activates cAMP-dependent protein kinases) and ascorbic acid (AA) for a minimum of 5 weeks (blue horizontal bars). During this differentiation process, specific protein surface treatments are used in different stages to ensure cell expansion and retainment of stem cell identity or differentiation (pink and light orange bars). The basic growth and differentiation media are a 1:1 mixture of DMEM/F12 and Neuralbasal media, including N2 and B27 (orange horizontal bar), and extra factors are added at different time points (modified from^[78,79])