Human integration-free iPS cells cells were maintained on growth factor reduced matrigel (BD) in mTesR1 medium (STEMcell Techonologies). Cells were passaged every 3-4 days with accutase (Millipore) and replated with mTesR1 supplemented with 10µM Y-27632 (Millipore), with media daily media change. To initiate differentiation, cells were plated at a density of 1.04x104 cells/cm2 on growth factor reduced matrigel in N2B27-CDM (DMEM/F12 medium containing 1% N2 supplement, 2% B27 supplement, 0.05% BSA fraction V, 1% Glutamax, 1% MEM-NEAA, and 110µM 2-mercaptoethanol (Life Technologies), supplemented with 10µM Y-27632 and 20ng/mL ß-FGF (Peptrotech). Twenty-four hours later, N2B27-CDM was replaced without Y-27632. Once cells reach approximately 60% confluency, cells were treated with mixture of KSR:N2 media (KSR: KO DMEM medium containing 15% knockout serum replacement, 1% glutamax, 1% MEM-NEAA, 55µM 2-mercaptoethanol; N2: DMEM/F12 medium containing 0.15% glucose, 1% N2 supplement, 20µg/mL insulin, 5mM HEPES) (Life Technologies) supplemented with 50nM LDN-13189 (Stemgent) and 5µM SB431542 (Tocris) on day 1 (100:0), day 2 (75:25), day 3 (50:50), day 4 (25:75), and day 5 (0:100). Twenty-four hours later, NC cells were harvested with accutase or TryplE (Invitrogen) and prepared for analyses or maintenance on growth factor reduced matrigel in SFM media (KO DMEM/F12 medium containing 2% StemPro Neural supplement and 1% glutamax) (Life Technologies) supplemented with 20ng/mL ß-FGF and 20ng/mL EGF (Sigma). To passage NC cells, cells were rinsed once with PBS and treated with accutase for 3 minutes. Cells were collected, pelleted, and re-plated at a density of 3x104 cells/cm2 on growth factor reduced matrigel (BD), fibronectin (Roche) or CellStart (Invitrogen) and grown in either N2 or SFM. When cells were 80-90% confluent, they were passaged.
Extracted molecule
total RNA
Extraction protocol
Total RNA from biological triplicates of healthy donor fibroblast reprogrammed iPS cells and differentiated NC iPS cells were extracted with Trizol and prepared for hybridization to Human Gene 1.0 ST Affymetrix microarrays using ~1 µg of starting material.
Label
biotin
Label protocol
According to manufacturer's (Affymetrix) recommendations
Hybridization protocol
According to manufacturer's (Affymetrix) recommendations
Scan protocol
According to manufacturer's (Affymetrix) recommendations
Description
Integration-free IPS cells were derived from a single healthy donor fibroblast. Replicates were obtained from independent cell culture wells.
Data processing
Data were analyzed in AltAnalyze version 2.0.8 (http://altanalyze.org) using RMA (bundled Affymetrix Power Tools). Ensembl gene-level estimates were derived using the mean expression of all AltAnalyze constitutive annotated probesets (database: EnsMart65). probe group file: HuGene-1_0-st-v1.r4.pgf
