H1 and CA1 human pluripotent stem cells (hPSCs) were routinely cultured on mitomycin C treated mouse embryonic fibroblast feeder cells in KSR media with 10ng/ml FGF2. KSR media contains 80% DMEM-F12 (GIBCO), 20% Knockout serum replacement (GIBCO), 0.1mM 2-mercaptoethanol, 2mM Glutamax, 0.1mM Non-essential amino acids. Prior to neural differentiation hPSCs were transferred to geltrex (GIBCO) coated surface in the absence of feeders and were cultured in mTESR2 (Stem cell technologies) media. On the day of neural induction, hPSCs were replated on geltrex coated surface in N2B27 media supplemented with 10µM SB431542 and 100nM LDN193189 and 10µM Y27632. Neural cultures were detached from Geltrex first on day 8-10 with Accutase or PBS-EDTA, and replated on laminin (Sigma) coated surface, in neural maintenance media (NES) supplemented with 10µM Y27632 at 3x105 cells per cm2 and 6F : 10ng/ml FGF2, 3μM CHIR99021, 1μM SB431542 (Peprotech), 100nM LDN193189, 100nM K02288 (Selleckchem), 100nM AKTiVIII, 75nM MK2206 (Selleckchem), and 1 μM XAV939 (Selleckchem). NES media contains 50% DMEM-F12, 50% Neurobasal, 0.1mM 2mercaptoethanol, 2mM Glutamax, 1x N2 supplement, 0.05x B27 minus vitamin A supplement (GIBCO). 4F media is made by supplementing NES media with 10ng/ml FGF2, 3μM CHIR99021, 1μM SB431542 or 50ng/ml FST (Peprotech), 100nM LDN193189 or 50 ng/ml NOGGIN. 6F media contains components of the 4F media with 100nM K02288 (Selleckchem), 100nM AKTiVIII and 75nM MK2206 (Selleckchem), and 1-2 μM XAV939 (Selleckchem). XAV939 concentrations need to be adjusted to each cell line, 1μM for H1, CA1, H9, CTRL hPSCd-erived cNESCs, 2 μM for Shef6 derived cNESCs. For single cell clone isolations, H1 (passage 12) cNES cells were cultured in 4F or 6F medium as described in section ‘Neuroepithelial stem cell maintenance and differentiation’. Three to four days after plating the cells were rinsed with PBS once and detached with 5-minute Accutase digestion at 37°C. The cells were collected with 10fold volume DMEM-F12 with 0.1% BSA and centrifuged at 300xg for 3.5 minutes. The pelleted cells were resuspended in 1 ml fresh 6F media and triturated by pipetting with 1000μl tip for 20 times with moderate force without generating bubbles. Cells were centrifuged with additional 5fold volume DMEM-F12 with 0.1% BSA at 300xg for 3.5 minutes. For single cell cloning cNES cells were plated in 6F media at 100 cells per cm2 culture surface area in a 10 cm dish coated with poly-D-lysine and laminin. FGF was added daily at 20ng/ml concentration without media change for the first 4 days. Media was completely changed after 6 days. Colonies were manually picked at day 10 to 96 wells. CA1 and H1 hESCs were also differentiated to dorsal forebrain neuroepithelium for 8-10d without culture in 6F condition. Mid-hindbrain specified NESCs (mhbNESCs) were derived in FGF and EGF from hPSCs (AF22, 23) or from human embryos (SAI1,3).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cultured cells with Qiagen RNeasy kit according to manufacturer’s instructions and was assessed for quality and quantity on a Bio-analyzer
Label
biotin
Label protocol
Purified RNA was labeled for analysis on Affymetrix Human Transcriptome Array 2.0 (Affymetrix) according to the manufacturer’s instructions, at Microarray Facility of The Centre for Applied Genomics at Sickkids, Toronto, Ontario.
Hybridization protocol
Purified and labeled RNA was prepared for analysis on Affymetrix Human Transcriptome Array 2.0 (Affymetrix) according to the manufacturer’s instructions, at Microarray Facility of The Centre for Applied Genomics at Sickkids, Toronto, Ontario.
Scan protocol
Prepared RNA was analyzed on Affymetrix Human Transcriptome Array 2.0 (Affymetrix) according to the manufacturer’s instructions, at Microarray Facility of The Centre for Applied Genomics at Sickkids, Toronto, Ontario.
Description
SAI3_32
Data processing
RMA method normalization, background substraction, and summarization was performed using Oligo (v1.34.2) R package from bioconductor. Normalized gene expression intensities were log2-scaled for all subsequent analyses. Unless otherwise stated, all data presented are representative of at least two independent experiments. A linear model approach and the empirical Bayes statistics were implemented for differential gene expression analysis, as described in limma (v3.26.9) R package user guide. P-values were adjusted using the Benjamini–Hochberg method and significance cut-off was set at 0.01.
