cell type: mesenchymal stromal cells from human bone marrow age: 21 year old human donor cell passage: 11
Biomaterial provider
University of Heidelberg
Treatment protocol
Cells were harvested by trypsination after passage 11.
Growth protocol
Human bone marrow samples were taken from the iliac crest (BM) of healthy donors . All samples were taken after written consent using guidelines approved by the Ethic Committee on the Use of Human Subjects at the University of Heidelberg. The mononuclear cell fraction was isolated by ficoll density fractionation and then taken into culture. The culture conditions have been first described by M. Reyes and colleagues (Reyes et al., 2001, Blood, 98, 2615-2625) and in our previous work (Wagner et al., 2005, Exp Hematol, 33, 1402-1416; Wagner et al., Exp Hematol, 34, 536-548). The medium consists of 58% Dulbecco´s Modified Eagles Medium-Low Glucose (DMEM-LG, Cambrex, Apen, Germany) and 40% MCDB201 (Sigma, Deisenhofen, Germany), 2% FCS (HyClone, Bonn, Germany), supplemented with 2 mM L-Glutamine, 100 U/ml Pen/Strep (Cambrex), 1% insulin transferrin selenium, 1% linoleic acid bovine serum albumin, 10 nM dexamethasone, 0.1 mM L-ascorbic-acid-2-phosphate (all from Sigma), PDGF-bb and EGF (10ng/ml each, R&D Systems, Wiesbaden, Germany). Tissue culture flasks were coated with 10 ng/ml fibronectin (Sigma) before use. MSC were cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide with medium changes twice a week. After 7-10 days initial colonies were replated in a new culture flask (passage 1, P1). Upon subconfluent growth (70%) cells were always replated at a density of 10(4) cells/cm(2).
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA of 10(6) MSC was isolated using the QIAGEN DNA Blood Midi-Kit. DNA quality was assessed with a NanoDrop ND-1000 spectrometer (NanoDrop Technologies, Wilmigton, Del) and average fragment length was assessed by gel electrophoresis.
Label
Cy3 and Cy5
Label protocol
Genomic DNA (500 ng) from each sample was bisulfite converted using the EZ-96 DNA Methylation Kit (Zymo research Corporation, Orange, US). This step leads to the deamination of non-methylated cytosines to uracils, while methylated cytosines are refractory to the effects of bisulfite and remain cytosine. After bisulfate conversion each sample was whole genome amplified and enzymatically fragmented.
Hybridization protocol
The HumanMethylation27 BeadChip (Illumina, San Diego, USA) was hybridized with 200ng DNA.
Scan protocol
The HumanMethylation27 BeadChip (Illumina, San Diego, USA) was scanned using the BeadArray Reader.
Description
alternatively named mesenchymal stem cells MSC-BM_21yrs
Data processing
Initial data analysis was performed with the BeadStudio Methylation software from Illumina (ie, BeadStudio Methylation Analysis Module 3.2.0). Raw data were background normalized.
