Fig. 5. Representative scheme illustrating the generation of committed DA neuron from hiPSCs, the DA neuronal differentiation by NPs treatments, and the analyses performed.

A hiPSCs obtained from two different healthy donors (Donor A and Donor B) were used. Neural induction (day 1–12) and dopaminergic commitment (day 12–30) have been obtained as previously reported [59], and described in the Materials and Methods section. Complete dopaminergic neuronal differentiation was reached at day 43 of culture in presence of cAMP. B At day 30, partially committed DA populations from both donors A and B were maintained in cAMP-free culture medium and treated, at day 31 (Step 1, analyzed at day 32) or at day 38 (Step 2, analyzed at day 39), for 24 h with 100 nM of NPs. The NPs treatments have been performed at day 31 or at day 38 for assessing if the effects produced could be dependent on the state of intrinsic differentiation of the committed neurons. Cultures maintained in cAMP- and NPs-free culture medium from day 30 to day 43 (Step 3) was used as control of cells grown in absence of factors inducing DA neuronal differentiation.