Neural progenitor cells (NPCs) were generated and cultured as previously described (Hargus et al., 2014; Reinhardt et al., 2013). Briefly, iPSC colonies were detached from MEFs 3–4 days after splitting using 2 mg/ml collagenase IV. Pieces of colonies were collected by sedimentation and resuspended in human ESC media (without FGF2) supplemented with 10 μM SB-431542 (Ascent Scientific), 1 μM dorsomorphin (Tocris), 3 μM CHIR 99021 (Axon Medchem), and 0.5 μM purmorphamine (PMA; Alexis) and subsequently cultured as embryoid bodies (EBs) in Petri dishes. On day 2, media was changed to N2B27 media containing the same small-molecule supplements. N2B27 medium consisted of Dulbecco’s modified Eagle’s media (DMEM)-F12 (Invitrogen)/Neurobasal (Invitrogen) 50:50 with 1:200 N2 supplement (Invitrogen), 1:100 B27 supplement lacking vitamin A (Invitrogen) with1%penicillin/streptomycin/glutamine (PAA). On day 4, SB-431542 and dorsomorphin were withdrawn and 150 mM ascorbic acid (AA; Sigma) was added to the media. On day 6, EBs were triturated into smaller pieces and plated on Matrigel- coated (Matrigel, growth factor reduced, high concentration; BD Biosciences) 12-well plates (Nunc) in NPC expansion media (N2B27 with CHIR, PMA, and AA). Media was changed every other day and cells were typically split 1:10–1:15 every 5–6 days. For splitting, cells were digested to single cells for approximately 10 min at 37C using prewarmed Accutase (PAA). Cells were diluted in DMEM (PAA) and spun down at 1,000 rpm for 5 min. The cell pellet was resuspended in fresh NPC expansion media and plated on Matrigel-coated cell culture dishes.
Extracted molecule
total RNA
Extraction protocol
RNA from microdissected human nigral tissue or iPSC-derived neurons was extracted using RNeasy columns (QIAGEN)
Label
biotin
Label protocol
GeneChipTM Human Gene 2.0 ST microarray procedure
Hybridization protocol
Standard Affymetrix protocol
Scan protocol
Standard Affymetrix protocol
Data processing
Human Gene 2.0 ST CEL files were normalized to produce gene-level expression values using the implementation of the Robust Multiarray Average (RMA) in the affy package (version 1.36.1) (Gautier et al., 2004) included in the Bioconductor software suite (version 2.11) (Gentleman et al., 2004) and an Entrez Gene- specific probeset mapping (17.0.0) from the Molecular and Behavioral Neuroscience Institute (Brainarray) at the University of Michigan (Dai et al., 2005). Array quality was assessed by computing Relative Log Expression (RLE) and Normalized Unscaled Standard Error (NUSE) using the affyPLM package (version 1.34.0).
