tissue: Placenta villous explant trimester: First Trimester
Growth protocol
Placental tissues were collected from consenting healthy women undergoing elective pregnancy termination at gestational ages between 7-9 weeks. dNK were isolated from decidua after collagenase and hyaluronidase digestion, depletion of attached cells, and were then enriched by human NK cell enrichment cocktail following the manufacturer’s instructions. The percentage of CD56+/CD16- dNK reached ~87%, CD16+ cells were < 2.1% (Hu et al., 2006). The dNK were trapped inside of hollow fiber (Spectrum Laboratories, Inc., Rancho Dominquez, CA, USA) (HF-dNK) and used for co-culturing with explants in order to eliminate trophoblast cell contamination from dNK when performing EVT DNA extraction. HF permits soluble factors with molecular weight below 500 kDa to be exchanged freely. For each placental specimen, villous explant cultures were set up in 12 well plates on collagen gel. After overnight incubation, explant cultures with EVT outgrowth on villous tips were identified and exposed to medium control (DMEM/F12 supplemented with 10% FBS), or IL-15 alone, or concordant HF-dNK plus IL-15 for a total of 96 hours. 6-8 explants were used for each treatment group in each placental specimen.
Extracted molecule
genomic DNA
Extraction protocol
For each of the explant cultures, EVT were harvested from collagen gel after dissecting villi tissue and pooled from each treatment group. Previous study has shown that the EVT forming the cell column and migrating on collagen were cytokeratin positive and vimentin negative cells and purity was high (Hu et al., 2006; Hu et al., 2008). Five placental specimens in total were used for global DNA methylation array and Bisulfite pyrosequencing validation. Genomic DNA from EVT was purified using QIAamp DNA mini kit according to the manufacturer’s instructions (Qiagen Inc. Mississauga, ON, Canada).
Label
Cy5 and Cy3
Label protocol
Cy5 and Cy3
Hybridization protocol
Illumina HumanMethylation450 BeadChip Protocol
Scan protocol
Arrays were imaged using Illumina iScan on a two-color channel
Data processing
GenomeStudio 2011.1 software and R version 2.14.0; Normalization using SWAN; The file Unmethylated_and_methylated_signal.txt is available on the series record and contains the raw data.
Genome-wide DNA methylation identifies trophoblast invasion-related genes: Claudin-4 and Fucosyltransferase IV control mobility via altering matrix metalloproteinase activity
Data table header descriptions
ID_REF
VALUE
Average Beta Values after normalization using SWAN
