cell type: iPSCs derived from human cord blood CD34+CD45+ cells
Growth protocol
Human induced pluripotent stem cells were cultured on Matrigel [BD Biosciences] in MEF conditioned medium (MEF-CM) supplemented with 8 ng/ml bFGF [Invitrogen] or maintained on irradiated feeder MEFs (iMEFs) with hPSC medium (DMEM/F12 [Gibco] with 20% Knockout Serum Replacement [Gibco], 100 mM b-mercaptoethanol, 100 mM nonessential amino acid [Gibco], 1 mM L-glutamine [Gibco], and 10 ng/ml bFGF, as previously described (Werbowetski-Ogilvie et al., 2009). Cells were routinely passaged every 5-7 days by mechanical transfer using 1 mg/ml Collagenase IV [Invitrogen]. For the derivation of naïve hPSCs, cells in primed pluripotent states were switched into hPSC medium supplemented with 1 mM MEK inhibitor PD0325901, 3 mM GSK3 inhibitor CHIR99021, and 10 ng/ml LIF (abbreviated LIF/2i) as previously reported (Buecker et al., 2010; Hanna et al., 2010; Li et al., 2009) or de novo reprogramming of somatic cells in naïve conditions. We also established naïve hPSCs using 10 ng/ml LIF and a combination of inhibitors for MEK/ERK (1 mM PD0325901), GSK3 (0.3 mM CHIR99021), LCF/SRC (1 mM WH-4-023), BRAF (0.5 mM SB590885) and ROCK (10 mM Y-27632) (5i)(Theunissen et al., 2014). Emerging colonies with a mESC-like morphology (appeared after 10-20 days of culture) were individually picked and subcultured by trypsinization or mechanical passaging onto feeder-free Matrigel or iMEFs with LIF/2i
Extracted molecule
total RNA
Extraction protocol
Extraction of total RNA using total RNA purification kit (Norgen Biotek, Canada) was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
In accordance with manufacturer's protocols
Hybridization protocol
In accordance with manufacturer's protocols
Scan protocol
GeneChips were scanned using Affymetrix® Scanner 3000 7G.
Data processing
Data was normalized using Robust Multichip Averaging (RMA) algorithm using Partek Genomics Suite 6.6 software.
