Stempro medium

All cells were cultured at 37° C under 5% CO2. U2987MG, a human glioma cell line [26 (link)], was cultured in 10% FBS-containing MEM supplemented with 4mM L-glutamine, 100units/ml penicillin and 0.1mg/ml streptomycin. The U2987MG cell line was established from a patient with high-grade glioma [26 (link)] and was denoted as primary culture 18. This cell line have then been very well studied [27 (link), 28 (link)] and subsequently established as a stable glioma cell line. U3016MG, U3047MG, U3117MG, U3065MG, U3060MG, U3062MG and U3101MG cells were cultured on laminin (10mg/ml) in serum-free (stem cell) conditions, to enrich for stem-like glioma cells, as described previously [29 (link)].
The human MC line LAD2 (obtained from Prof Dean Metcalfe at NIH/NIAID, MD, USA) was cultured as described previously [30 (link)] in StemPro medium supplemented with 4mM L-glutamine, 100units/ml penicillin and 0.1mg/ml streptomycin and 100ng/ml SCF (Invitrogen, Carlsbad, USA).

The human HD-iPSC line (60 CAG HD line) from the Coriell Institute for Medical Research was cultured on L7 (Lonza) matrix in STEMPRO medium (Invitrogen) supplemented with 10 ng/mL recombinant human fibroblast growth factor 2 (FGF2). Cells were fed daily and manually passaged every 5–7 days. Striatal neuron precursors were derived as previously described in Nicoleau et al.⁶⁰ (link) and Arber et al.⁶¹ (link) Neuralized and patterned human iPSCs, enriched for ventral telencephalic progenitors, were collected after 21 DIV using Accutase for 10–20 min at 37°C and frozen in CryoStor CS10 (Invitrogen) freezing media. For terminal neuronal differentiation of striatal precursors, cells were plated on polyornithine laminin-coated dishes at 40,000 cells/cm² in DMEM/F12 media supplemented with N2, B27, 20 ng/mL brain-derived neurotrophic factor (BDNF; R&D Systems), 0.5 mM N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbcAMP; Sigma-Aldrich), 0.5 mM valpromide (Lancaster Synthesis), and 25 ng/mL Activin A for 30–35 additional days.

Six hES cell lines-H1 (WA01), H7 (WA07), H9 (WA09), and H14 (WA14) (WiCell, Madison, WI, USA) and CM7 and CM14 (established at Galat lab [23 (link)])-were used for this study. For the microarray analysis H7, H9, H14, CM7, and CM14 cells were grown in StemPro medium (Invitrogen, Carlsbad, CA, USA) on a Matrigel substrate (BD Bioscience, San Jose, CA, USA). The confluent cultures of hESCs growing on 10-cm dishes were split to 6 experimental 10-cm dishes mechanically by using the StemPro EZ Passage tool (Invitrogen). For amino acid assays, H9 cells were maintained in DMEM/F12 and supplemented with Knockout Serum Replacement (SR-1), FGF, 2,β-mercaptoethanol, and GlutaMax, Gibco Inc., Billings, MT, USA). For RNAseq analysis, H1 cells were maintained on Matrigel-coated culture dishes in StemMACS iPS-Brew XF (Miltenyi Biotec, Bergisch Gladbach, Germany). A single-cell suspension was counted with the help of an automatic cell counter, Nexcelom (SelectScience, Church Farm Business Park, UK), during cell lifting for the analysis. Cell confluence and morphology were observed daily.