Generation of human neurons in vitro and drug treatment

The long-term neuroepithelial stem cell (NESC) line I3.2, derived from human embryonic stem cells and previously described⁸² was used for the generation of human neurons in vitro. For maintenance, NESC were cultured on poly-l-Ornithine (PLO, Sigma-Aldrich, Irvine, UK) and laminin (Sigma-Aldrich, Irvine,UK) coated wells, in DMEM/F12 (Gibco) media supplemented with N2 and B27 supplements (Life Technologies; Carlsbad, CA, USA) in the presence of the growth factors EGF and FGF2-basic as previously described⁸². To induce differentiation, EGF and FGF2-basic were removed from the media (Supplementary Fig. ^{2A and B}). Briefly, NESC were seeded on PLO/laminin at the density of around 75000–150000 cells per well, in a 48-well plate, and cultured for 7 days in DMEM/F12 media supplemented with N2 (1:100) and B27 (1:100) supplements. After one week of differentiation, media was changed to DMEM/F12 supplemented with N2 (1:100) with the addition of glial cell-derived neurotrophic factor (GDNF) 20 ng/ml, brain-derived neurotrophic factor (BDNF) 20 ng/ml, ascorbic acid 10 mM, dibutyryl adenosine 3′,5′-cyclic monophosphate sodium salt (dcAMP) 25 mM. Drug treatments started after 2 weeks of in vitro differentiation, when NESCs had a clear neuronal morphology. Differentiated neurons were characterized through immunohistochemistry stains for neuronal marker TUBB3.

For drug treatments, neurons were treated in plain DMEM/F12 media supplemented with only N2 (1:100), with the addition of either the vehicle or different concentrations of the drugs for one week before they were processed for DNA extraction. Clozapine and risperidone were dissolved in 1 M HCl before added to cell culture medium in the concentrations of 0.075 μM, 0.75 μM and 0.025 μM, 0.25 μM respectively. The vehicle used was plain DMEM/F12 media acidified by 0.0025 μM HCl, pH neutralized by CO₂ buffer. The concentrations were selected to simulate clinical target concentrations in plasma and diluted by 10-fold to simulate concentration in the cerebrospinal fluid and brain interstitial fluid^83–85. At the end of treatment, cells were lysed and DNA extraction was performed using the Quick-DNA™ Miniprep Plus Kit (Zymo Research Corp, Irvine, CA, USA). Subsequently mtDNA copy number determination was performed as described above. Experiments were run in 3 biological replicates.