Institutional Review Board (DSRB, National Healthcare Group) approval was given for collection of umbilical cords from human subjects by informed patient consent. hWJSCs were derived from 6 different umbilical cords using a method previously described by our group (Fong et al. 2007. Reprod Biomed Online 15:708-718). Primary cultures of hWJSCs were established in sterile plastic tissue culture flasks (BD, USA) in the presence of a culture medium comprised of DMEM (high glucose), 20% fetal bovine serum (FBS), glutamine, insulin-transferrin-selenium (ITS), bovine fibroblast growth factor (bFGF) and antimycotic-antibiotic solution (Invitrogen, USA). Once confluent, the hWJSCs were passaged by detachment and disassociation with trypsin, washed and re-seeded into new flasks in the presence of the same medium above. A karyotypically normal human embryonic stem cell line (hESC) (HES3, ESI Singapore Ltd.) and a variant hESC line (BG01V, SCRC-2002, ATCC) were cultured on mitomycin-C treated mouse embryonic fibroblasts (MEFs) in bulk culture medium (BCM) composed of KnockOut DMEM medium (Invitrogen) supplemented with 20% KO serum replacement, 0.1 mM ß mercaptoethanol, 1 mM glutamine, 0.1 nM nonessential amino acids, 50 units /ml penicillin, 50 µg/ml streptomycin, 1:200 dilution of ITS (insulin-transferrin-selenium) (Invitrogen) and 16 ng/ml bFGF (Chemicon). Three human embryonic carcinoma cell lines (hECC) (NCCIT, CRL-2073, ATCC), (NTERA-2 cl.D1, NT2D1; CRL-1973, ATCC), and GCT27C4 (kind gift from Dr Martin Pera) were cultured respectively in the following basal media RPMI 1640, High Glucose DMEM, and DMEM/F12 (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone) and L-glutamine (Invitrogen). A human fetal fibroblast cell line (hFC) (Detroit 551, D551; CCL-110, ATCC) was grown in DMEM supplemented with 10% fetal bovine serum (Hyclone) and L-glutamine (Invitrogen).
Extracted molecule
total RNA
Extraction protocol
Total RNA from the hESC and hWJSC cell lines were extracted with RNeasy Mini Kit (Qiagen). Total RNA from the hECC and human fibroblast cell lines was prepared according to the TRIzol reagent protocol (Invitrogen) and purified with the RNeasy MinElute Cleanup Kit (Qiagen). Total RNA yield was determined using Nanodrop™ 1000 (Thermo Fisher Scientific) while RNA integrity was evaluated with Bioanalyzer 2100 (Agilent Technologies).
Label
biotin
Label protocol
The microarray experiment was carried out according to the manufacturer’s recommendations for the Affymetrix GeneChip Human Genome U133 plus 2.0 Array. The GeneChip One-Cycle cDNA Synthesis Kit (Affymetrix) was used to perform the first-strand and second-strand synthesis reactions. First-strand synthesis was carried out with 3.5 µg sample RNA, poly-A RNA controls (Eukaryotic Poly-A RNA Control Kit, Affymetrix), and T7-Oligo(dT) Primer at 42 oC for 1 hr. The second-strand reaction was performed at 16 oC for 2 hrs. The double-stranded cDNA was purified with the GeneChip Sample Cleanup Module (Affymetrix). In vitro transcription to produce biotin-labeled cRNA was carried out with the GeneChip IVT Labeling Kit (Affymetrix) at 37 oC for 16 hrs. The adjusted cRNA yield was determined by the difference between the amount of cRNA measured after in vitro transcription and the starting amount of total RNA. 20 µg of purified biotin-labeled cRNA was then fragmented at 94oC for 35 min.
Hybridization protocol
The GeneChip Hybridization, Wash and Stain Kit (Affymetrix) was used for the hybridization, wash and scan steps. 15 µg of fragmented cRNA probes were mixed with the Control Oligonucleotide B2 and Eukaryotic Hybridization Controls (bioB, bioC, bioD, cre) before application to the GeneChip Array. Hybridization was carried at 45 oC, 60 rpm for 16 hrs in the Hybridization Oven 640 (Affymetrix). Washing of the GeneChip Arrays was done on the Fluidics Station 450 (Affymetrix) with the Fluidics Script FS450_0001.
Scan protocol
Scanning was carried out with the GeneChip Scanner 3000. The scanned data was retrieved using the Affymetrix GeneChip Operating Software (GCOS) and inspected with the quality control parameters for image quality, presence of artifacts, grid alignment, border staining and hybridization controls.
Description
NCCIT_R2
Data processing
The Affymetrix GeneChip Human Genome U133 plus 2.0 Array CEL files were then imported into Expression Console (Affymetrix) for data processing. Quality control parameters were checked for the tabular report, signal distribution, and array comparisons. The performance report and signal detection calls for each GeneChip Array were analysed with the MAS 5.0 analysis. The expression summary file was generated with the RMA algorithm (parameters were RMA Background Correction to PM-only probes and quantile normalization). Two datasets analyses were performed. The first dataset contained the hESCs, hECCs, hWJSCs and hFC samples that were generated by our group. The second dataset contained a meta-analysis of the hWJSCs and hFC that were generated by our group and selected samples available from the public GEO database (human mesenchymal stem cells, umbilical vein endothelial cells, normal stroma, tumour stroma, fibroblast ).
