Differentiation of hES cells line VUB01 towards neural rosettes Neural precursor phenotype
Growth protocol
GROWTH PROTOCOL (hES): hES cells were maintained and propagated on a feeder layer of STO (SIM mice Thioguanine and Ouabaine resistant) murine embryonic fibroblast cell line inactivated by Mitomycin C (Sigma Aldrich, 2.5 μg per mL overnight at 37°C). Cells were cultured within a humidified 10% CO2 incubator setted at 37°C in a serum replacement medium (DMEM, 20 % Knock-out Serum Remplacement (KSR), 1% Glutamax 1mM, 1% Non Essential Amino Acid (NEAA), 0.1% Beta-Mercaptoethanol (BM) 0.1% and 1% Penicillin/streptomycin (P/S), all from GIBCO) supplemented with 8ng/mL of bFGF (Invitrogen). DIFFERENTIATION PROTOCOL (NPC): The protocol for the differentiation of hES towards neurectodermal rosettes was adapted from (Perrier at al). Briefly, hES cells were dissociated from STO by manual dissociation and plated in a density of approximately 103 cells per cm² on a confluent layer of mitotically inactivated murine stromal feeder (MS5). The cells were cultured in KSR medium (Knock-out Serum Replacement, 15 % SR; 1% Glutamax; 1% NEAA and 0.1% BM, all from GIBCO) for 14-16 DIV (Days in Vitro), replaced by Neurobasal medium, N2 (DMEM-F12 + Glutamax, 1% N2 supplement and 1% P/S) until DIV21. Cells were harvested at DIV21 using TrypLE Express (GIBCO) and about 5.106 cells were suspended in PBS -2% Fetal Calf Serum containing 1% of 7-amino-actinomycin D (7AAD) (Sigma) and then incubated with IgG1κ Direct conjugated Phyco-erythrin (PE) monoclonal anti human Neural Cell Adhesion Molecules (hNCAM) antibody diluted 1/10 provided by BD Biosciences Pharmingen™. This antibody recognizes an extracellular immunoglobulin-like domain common to three molecular weight forms –Mr 120, 140 and 180 kilodaltons –of the NCAM protein. The cell sorting was performed by MoFlow Cell Sorter Cytometer from Cytomation and alived positive and negative fractions were collected in 1mL of N2 medium with 1% P/S. DIFFERENTIATION PROTOCOL (MSC): Mesodermal differentiation was obtained based on a protocol described by Barberi et al Briefly, differentiation was induced by plating 2.104 ES cells/cm2 on 0.1% gelatin coated dishes in the presence of KO-DMEM medium supplemented with 20% of Fetal Bovine Serum (FBS, Invitrogen), 1mM L-glutamine, 1% non-essential amino-acid, 1% penicillin-streptomycin and 0.1mM β-mercaptoethanol. Medium was changed every other day. Confluent cells were passed with trypsin/EDTA 1X (Invitrogen) in new gelatin coated dishes.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the cells using the RNeasy Mini kit (Qiagen) according to the manufacturer protocol
Label
Biotin
Label protocol
100 ng of total RNA was used to generate biotinylated cRNA using the GeneChip® Two-Cycle Target Labeling and Control Reagents from Affymetrix according to the manufactures protocol
Hybridization protocol
Hybridisation, washing and staining procedures of the Affymetrix GeneChip® Human Genome U133 Plus 2.0 were carried out as described in the Affymetrix technical manual (see manufacturer's web site)
Scan protocol
Scanning procedures of the Affymetrix GeneChip® Human Genome U133 Plus 2.0 were carried out as described in the Affymetrix technical manual (see manufacturer's web site)
Description
gene expression data for VUB01 hES cells line-derived NPC
Data processing
Hybridizing data were exploited using Array Assist 4.2 software (Stratagene). First, the software allowed validating quality controls. Next, experiment grouping was performed and GC-RMA statistical algorithm procedure was used to normalize hybridization intensity values. After this step, we used one way-ANOVA test on transformed logarithm basis 2 data to retain only values were not changing significantly (α<0,05) among triplicate sample. Next, we compared on the one hand NCAM-NPC to ES and on the other hand CD73-MPC to ES using Student parametric statistical test adjusted by FDR Benjamini-Hodgberg correction. At last, we retained as significant modulated genes those that at last one probesets vary to up to 2 folds (Fold Change, FC >2) with a corrected p-value, αc<0,05. The final gene lists was established by removing doubling datas (several probesets measured the same gene) to retain the most modulated one. At last, we also performed the correlation plot between the 18 arrays of the experiment using this software.
