MSCs were isolated from bone marrow (tibia plateau) after written consent using guidelines approved by the Ethic Committee of the Use of Human Subjects at the University of Aachen (permit number: EK128/09). MSCs from three donors were infected with pMXs based retroviruses (Addgene, Cambridge, MA, USA) carrying the OCT3/4, SOX2, KLF4, and c-MYC genes. Established iPSCs were maintained on MEFs in DMEM/F12 medium supplemented with Glutamax, 20% knockout serum replacer, 1% nonessential amino acids, 1x penicillin/streptomycin, 1x L-glutamine, 0.1 mM β-Mercaptoethanol, and 50 ng/ml basic fibroblast growth factor (Peprotech, Hamburg, Germany). To exclude contamination of feeder layer cells, iPSCs were adjusted to a feeder-free system on matrigel (BD Biosciences, San Jose, CA, USA) in mTeSR™1 medium (Stemcell Technologies, Vancouver, BC, Canada) for at least three passages. Cells were passaged every 5-6 days enzymatically with dispase (1 g/ml, Stemcell Technologies). For re-differentiation of iPSC towards iPS-MSCs medium was simply exchanged for MSC standard medium supplemented with 10% human platelet lysate (hPL) for 7 days, and cells were then further passaged in culture wells coated with 0.1% gelatin (Sigma-Aldrich, St. Louis, CA, USA).
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was isolated using the Qiagen DNA Blood Midi Kit.
Label
Cy5 and Cy3
Label protocol
Standard Infinium HD Methylation Assay protocol
Hybridization protocol
Bisulfite converted DNA was amplified, fragmented and hybridized to Illumina Infinium HumanMethylation450 Beadchip using standard Infinium HD Methylation Assay protocol.
Scan protocol
Standard Infinium HD Methylation Assay protocol
Data processing
Initial analysis was performed by the GenomeStudio 2010.3 (Modul M Version 1.8.5). Data were normalized with internal controls according to Illumina´s standard procedures. Methylation level at each locus was calculated with the GenomeStudio Methylation module as beta-value (ranging from 0 to 1). The number of beads per feature varies between chips and beta-values were calculated as average of at least three technical replica.
Tissue- and Aging-specific DNA-Methylation Patterns are erased in Mesenchymal Stromal Cells derived from Induced Pluripotent Stem Cells. [Methylation profiling]
