Human iNeurons

All the iPSC lines used in the study express doxycycline-inducible Ngn2 and were used to generate homogeneous iPSC-derived glutamatergic neurons using a previously described protocol (25). iPSCs were singularized using StemPro Accutase Cell Dissociation Reagent (Gibco #A11105-01), neutralized with mTESR1 media, centrifuged, and pelleted at 200g for 5 min. The pelleted cells were resuspended in induction media [IM recipe: KO DMEM/F12 (Gibco #12660-012), 1× NEAA, 1× N2 supplement (N2; Gibco #17502-048), NT-3 (10 ng/ml; PeproTech #450-03), brain-derived neurotrophic factor (BDNF) (10 ng/ml; PeproTech #450-02), mouse laminin (1 μg/ml; Thermo Fisher Scientific #23017-015), 10 nM Y27632 (ROCK inhibitor; Tocris #125410), and doxycycline hydrochloride (2 μg/ml; Sigma-Aldrich #D9891)], counted, and 7 × 10⁵ cells were plated onto a Matrigel-coated well of a six-well plate in 2 ml of IM or 4.5 × 10⁶ cells onto a 10-cm dish. Medium was changed every day with fresh supplements.

Three days after doxycycline induction, the cells were released with StemPro Accutase, centrifuged, pelleted, and resuspended in maturation media [MM recipe: half DMEM/F12 and half Neurobasal-A (Gibco #10888-022), 1× NEAA, 0.5× GlutaMAX (Gibco #35050-061), 0.5× N2, 0.5× B27 supplement (Gibco #17504-044), NT-3 (10 ng/ml), BDNF (10 ng/ml), mouse laminin (1μg/ml), 10 nM Y27632, and doxycycline hydrochloride (2 μg/ml)], counted, and 5 × 10⁵ cells were plated onto freshly prepared poly-l-ornithine/laminin–coated wells of a 12-well culture dish or 11.5 × 10⁶ cells onto a 15-cm dish. Some iPSC lines with individual gene KDs exhibited KD-related differences in the growth and survival of iPSCs/iNeurons. The same number of cells were plated for these iPSC cell lines during replating to avoid any density-related differences in differentiation or αSyn expression. The cells that were replated after 3 days of induction were termed “day 0 iNeurons.” Half of the media was removed and replaced with new MM media with fresh supplements (minus doxycycline) on day 7. All media changes were made without doxycycline after day 7. All media changes were made without doxycycline and Y27632 after day 7. Half of the media was removed and replaced with new MM media with fresh supplements (minus doxycycline and Y27632) on days 7 and 14. One-third of the media was removed and replaced with twice that volume with fresh supplements on day 21. One-third of the media was removed and replaced with the same volume of media on day 28.