Stemflex

iPSCs were maintained in Stemflex (Gibco # A334901) and mTeSR Plus (STEMCELL Technology # 100-0276) medium in feeder-free conditions on a basal matrix of Matrigel (Corning # 47743-706). ReLeSR (STEMCELL Technologies # 100-0484) was used for routine maintenance splitting.

Human embryonic stem cells H9 [Wisconsin International Stem Cell (WISC) Bank, WiCell Research Institute, WA09 cells] were cultured according to WiCell stem cell protocols in 6-well plates on Matrigel (Corning, hESC-Qualified) in StemFlex (Gibco). Use of stem cell line H9 was approved by the Steering Committee of the UK Stem Cell Bank and for the Use of Stem Cell Lines (ref: SCSC18-05).
Primary mixed glial cultures were derived from P0-P2 neonatal Spraque Dawley rats and were generated along the previous guidelines [80 (link)], with minor modifications [81 (link)]. The pups were euthanized following Schedule 1 rules and regulations from the Home Office Animal Procedures Committee UK (APC). Mixed glia cells were maintained for 10 days in culture after which flasks were shaken for 1h at 260rpm on an orbital shaker to remove the loosely attached microglia, and then overnight at 260rpm to dislodge oligodendrocyte precursors. Astrocyte cultures were then maintained in glial culture medium (Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), glutamine and 1% pen/strep) and passaged at a ratio of 1:3, every 10–14 days. Cells were passaged at least once before co-culturing with iNs and were only used between passages 2 and 5.

hPSC lines used in this study were obtained from WiCell (H1), the Allen Institute (Allen line), the Harvard Stem Cell Institute iPS Core (DiPS 1016 SevA). hPSC lines were maintained in feeder-free conditions in StemFlex (Gibco) media on Matrigel (Corning) in 6-well tissue culture dishes. For passaging, hPSCs were detached as clumps using Versene Solution (Thermo Fisher) and replated at a ratio of 1:8–1:10.

The iPSC lines were maintained in a serum-free and feeder-free system, as previously described [27 (link),28 (link)].Cardiac differentiation was instigated using the PSC Cardiomyocyte Differentiation Kit (Gibco, Waltham, MA, USA) according to the manufacturer’s instructions. In brief, undifferentiated iPSCs were seeded on 12-well plates coated with GelTrex (Gibco, Waltham, MA, USA) and cultured in StemFlex (Gibco, Waltham, MA, USA) media to 90–100% confluence. Afterward, the cells were cultured subsequently in medium A (first 2 days), medium B (next 2 days), and cardiomyocyte maintenance medium (for approximately 7–14 days). Beating human iPSC-CMs were dissociated with collagenase B and subjected to immunostaining using antibodies specific for cardiac troponin T or sarcomeric α-actinin [28 (link)]. To evaluate specific functional properties of interest, iPSC-CMs were treated with different drugs or transfected with siRNAs specific to CAV3 (S2454, Ambion, Waltham, MA, USA) or MeCP2 (AM16708, Ambion, Waltham, MA, USA).

iNeuron generation was based on previously described protocols (43 (link)). To increase genome integration, the mCherry, puromycin, and hNGN2 plasmids were moved into the PiggyBac system. Briefly, the parental WT control, and 2 independent clones of each I1061T-NPC1 and R934L-NPC1 iPSCs were plated on plates with coated Matrigel (Corning, 354277) in the presence of 1 μM Y-26732 (Cayman Chemical, 10005583) in StemFlex (Gibco, A3349401). On day 1, the media were changed to E8 (Stemcell, 05990) alone, and cells were transfected using 10 μL Lipofectamine Stem Transfection Reagent (Invitrogen, STEM00015) diluted in 125 μL Opti-MEM (Gibco, 31985062). 0.9 μg PBase, 0.7 μg mCherry, 0.7 μg hNGN2, and 0.2 μg puromycin plasmids were mixed in 125 μL Opti-MEM. On day 2, the media were changed to StemFlex. On day 3, cells were lifted into a single-cell dilution series using Accutase (Stem Cell, 07922); cells were added into several culture plates with StemFlex and Y-27632. On days 4–5, cells were fed with StemFlex. Antibiotic selection was performed on day 6; StemFlex media were replaced with StemFlex containing 0.85 μg/mL puromycin (Invivogen, ant-pr-1). Selection was observed on day 7–8. On day 9–14, 10 mCherry⁺ colonies were selected, and neuron differentiation was characterized to identify the most efficient clones.