Neurocult sm1 neuronal supplement

Single mammary gland cell suspensions were depleted of hematopoietic cells by incubation with anti-CD45 (70-0451, TONBO), followed by Dynabeads® Sheep anti-Rat IgG according to the manufacturer's instructions (Invitrogen, 11035). Depletion was confirmed by flow cytometry using APC-conjugated anti-CD45.2 (20-0454-U025, TONBO). Mammosphere assay was performed as described by Boyle et al. (13 (link)), with a few modifications. Briefly, 2 × 10⁴ freshly isolated CD45⁻ mammary gland cells were seeded in triplicates into 96-well ultra-low attachment plates (Corning Inc.) pre-coated with poly(2-hydroxyethyl methacrylate) (P3932, Sigma) in a 1:1 mixture of DMEM and Ham's F12 medium (Sigma) supplemented with NeuroCult SM1 Neuronal Supplement (05711, StemCell Technologies), 20 ng/ml bFGF (78003.1, StemCell Technologies), 20 ng/ml EGF (78006, StemCell Technologies), 4 μg/ml heparin (07980, StemCell Technologies), penicillin-streptomycin and fungizone. Mammosphere cultures were incubated at 37°C for 7 days. At the end point, mammospheres of at least 40 μm diameter were counted under the microscope at 40X magnification. Digital images were used to calculate mammosphere size using Image J software.

Dissociated hippocampal cultures were prepared from embryonic day (E)18 rat brains of both genders (Kapitein et al., 2010b (link)). Dissociated neurons were plated on Ø18-mm coverslips coated with poly-L-lysine (37.5 μg/ml, Sigma-Aldrich) and laminin (1.25 μg/ml, Roche Diagnostics) at a density of 100,000 neurons per well, in Neurobasal medium (NB), supplemented with 1% penicillin and streptomycin (pen/strep), 2% B27, and 0.5 mm L-glutamine (all from Invitrogen; NB-complete medium) at 37°C in 5% CO_2. From day in vitro (DIV) 1 onwards, medium was refreshed every 7 d by replacing half of the medium with Brainphys neuronal medium supplemented with 2% NeuroCult SM1 neuronal supplement (Stem Cell Technologies) and 1% pen/strep (BP-complete medium).

Lymphoblast cell lines were acquired from the Coriell Institute for Medical Research, Line # GM15010, female origin (New Jersey, USA) from a patient carrying a triplication in the SNCA gene, reprogrammed into induced pluripotent stem cells (iPSCs) called line ‘3x-1’, and characterized previously (Stojkovska et al., 2022 (link)). iPSCs were cultured on Matrigel (Corning, #354277) coated plates and maintained in mTESR1 media. Differentiation into midbrain dopaminergic neurons occurred using previously established protocols (Kriks et al., 2011 (link)). Briefly, iPSC lines were accutased (Corning, # 25058CI) and seeded onto Matrigel (Corning, #354277) coated plates, allowed to grow to confluency, then treated with dual SMAD inhibitors followed by a cocktail of growth factors (Cuddy et al., 2019 (link)). After differentiation, the neurons were cultured in neurobasal medium (ThermoFisher, # 21103049) with NeuroCult SM1 Neuronal Supplement (Stem cell technologies, #5711) and 1% glutamine and penicillin/streptomycin. Patient derived SNCA triplication IPSC-neurons were used to evaluate the effects of HKL in a human relevant model of synucleinopathy. Neurons were matured for 60 days and subsequently treated with 10 μM HKL or 0.01% DMSO for 72 h. Cells were pelleted, snap frozen, and shipped to Mayo Clinic-Jacksonville for αsyn and SNCA mRNA quantitation.

Primary hippocampal cultures were prepared from E18 Sprague-Dawley rat embryos according to the Banker protocol (Kaech and Banker, 2006). Hippocampi were dissected in Petri dishes filled with HBSS and HEPES, and dissociated by trypsin treatment (0.05%; Gibco) at 37 °C followed by trituration with flame-polished Pasteur pipettes. Cells were electroporated and plated at a density of 250,000 per 60-mm dish, on poly-L-lysine pre-coated 1.5H coverslips (Marienfield, cat. No. 117580), pre-plated with 75,000 non-electroporated cells. After 2 hours, coverslips were transferred to dishes containing an astrocyte feeder layer, plated at a density of 40,000 cells and cultured in MEM (Fisher scientific, cat No. 21090-022) containing 4.5 g/l Glucose, 2 mM L-glutamine and 10% horse serum (Invitrogen) for 14 days. Neuron cultures were maintained in Neurobasal medium supplemented with 2 mM L-glutamine and 1X NeuroCult SM1 Neuronal supplement (STEMCELL technologies) for 14–16 days.

Dissociated hippocampal cultures were prepared from embryonic day 18 (E18) rat brains of both genders, as described in^{42 (link)} and in accordance to the approved DEC work-protocol as mentioned in the ethics statements above. Mother rats were sacrificed by gradual fill CO2/O2. Subsequently the uterus containing the pups is taken out and is stored in a sterile ice cold environment. After the pups were sedated by the cold, they were removed from the uterus and decapitated. Dissociated neurons were plated on Ø18-mm coverslips coated with poly-L-lysine (37.5 µg/ml, Sigma-Aldrich) and laminin (1.25 µg/ml, Roche Diagnostics) at a density of 100,000 neurons per well. Neurons were grown in Neurobasal medium (NB) supplemented with 1% pen/strep, 2% [v/v] B27, and 0.5 mM L-glutamine (all from Gibco) (NB-complete medium) at 37 °C in 5% CO_2. From days in vitro (DIV) 1 onward, medium was refreshed weekly by replacing half of the medium with Brainphys neuronal medium (BP) supplemented with 2% [v/v] NeuroCult SM1 neuronal supplement (STEMCELL Technologies) and 1% pen/strep (BP-complete medium).