Isolation and culture of MSC
MSC were obtained from excessive material of diagnostic bone marrow aspirates of children with hematopoietic malignancies after informed consent and approval by the local IRB (241/2005V). MSC cultures were established as described earlier . Briefly, bone marrow samples (0.5 ml) were heparinized, subjected to red blood cell lysis using the ammonium chloride method, and washed with Hank's buffered salt solution (Lonza, Basel, Switzerland). Cells were placed in low glucose Dulbecco's Modified Eagle Medium (LG-DMEM, Lonza) supplemented with 5% (v/v) human fresh frozen plasma (FFP), 107/ml platelets (University of Tübingen blood donor centre), 80 IU/ml heparin sulphate, 1 mM glutamine (Lonza), 100 IU/ml penicillin, 100 μg/ml streptomycin (both Biochrom, Berlin, Germany) in one well of a six-well culture plate. Nonadherent cells were purged on day 2 and medium was replaced twice a week. At 80% confluence, cells were harvested with trypsin 0.5% (Lonza) and replated in the above medium at 2,000 cells/cm2. To expand the cells, successive passages were performed with the same protocol for a maximum of six weeks.
Equal numbers of culture dishes of the same donor were kept in Heracell gas addition incubators (Heraeus Instruments GmbH, Hanau, Germany) with gas mixtures consisting of either 21% O2, 74% N2, 5% CO2 (referred to as 21% O2), 1% O2, 94% N2, 5% CO2 (referred to as 1% O2) or 3% O2, 92% N2, 5% CO2 (referred to as 3% O2) for at least one week before performing an experiment.
After 7 days of adaptation in 21%, 3% and 1% O2, respectively, MSC were plated in a 96 well plate at a density of 6,250 cells/cm2 and cultured for another 7 days. Proliferation was analyzed using the MTS assay kit according to the instructions of manufacturer (Promega, Madison, WI).
Cell Cycle Analysis
Cell cycle analysis of MSC was performed after 7 days and 21 days of adaptation in 21% O2 and 1% O2. 106 MSC were washed with phosphate buffered saline (PBS, Biochrom, Berlin, Germany) three times, fixed with ice-cold 70% ethanol and stored at 4°C for a minimum of 1 hour. After three more washing steps with PBS, DNA and RNA of the MSC were stained with propidium iodide/RNase staining buffer (BD Biosciences, Heidelberg, Germany). The samples were incubated at room temperature for 15 minutes and analyzed by flow cytometry on a FACS Calibur (Becton Dickinson, Heidelberg, Germany).
The differentiation of MSC was assessed after an adaptation period of 14 days in 21% O2 and 1% O2 as reported earlier .
Adipogenic differentiation was induced in 80% confluent MSC cultures by supplementing the cell culture medium with 1 μM dexamethasone, 60 μM indomethacin, 0.5 mM isobuthylmethylxanthine (all Sigma-Aldrich, Steinheim, Germany) and 10 μM insulin (Novo, Novodisk, Bagsværd, Denmark). After 14 to 20 days, histochemical staining with Oil-Red-O (Sigma) was performed to detect lipid droplet formation.
Osteogenic differentiation was induced in 50% confluent MSC cultured in cell culture medium supplemented with 10 nM dexamethasone and 0.1 mM L-ascorbic acid-2-phosphate (both Sigma) from day 0 to day 7. For 7 to 14 more days, 10 mM beta-glycerol phosphate (Sigma) and 100 ng/ml bone morphogenic protein-2 (Tebu-Bio, Magenta, Italy) were added as additional cell culture supplements. To verify the osteogenic differentiation, calcium precipitates were detected by aqueous 0.5% (v/v) Alizarin Red-S (Sigma) histochemical staining. This differentiation was also performed at 3% O2.
MSC of the same donor were placed in 21% O2, 3% O2 or in 1% O2 atmospheres for 14 days. Subsequently, MSC were harvested, washed with PBS supplemented with 2% Fetal Calf Serum (FCS, Biochrom) and stained with the following antibodies: anti-IgG1-FITC (cloneMOPC-31C), anti-IgG1-PE (clone G18-145), anti-CD45-FITC (clone HI30), anti-CD34-PE (clone 563), anti-CD73-PE (clone A02), anti-HLA-DR-FITC (clone TÜ36), anti-HLA-ABC-PE (clone G46-2.6), all from Becton Dickinson and anti-CD105-FITC (clone N1-3A1, Ancell, Bayport, MN, USA), anti-CD90-PE (clone F15-42-1), anti-CD106-FITC (clone 1.G11B1, both Serotec, Düsseldorf, Germany) and anti-CD146-PE (clone P1H12 from Santa Cruz Biotechnology, Heidelberg, Germany). The immunophenotype was analyzed using a FACS Calibur and CellQuest analysis software (Becton Dickinson).
Array-based Comparative Genomic Hybridization (array-CGH, matrix-CGH)
Array- (or Matrix-) CGH  was carried out as previously described [40–42]. Selection of genomic clones, isolation of BAC DNA, performance of DOP-PCR, preparation of microarrays, labeling, hybridization and washing procedures were performed as outlined. Raw data processing and normalization was performed as previously reported providing log2-ratios of spot intensities . The chromosomal mapping information is based on the University of California at Santa Cruz (UCSC) genome database (March 2006) and the March 2006 (hg18) assembly (NCBI Build 36.1) of the International Human Genome Sequencing Consortium. All clones from totally 11 samples were subjected to pre-processing, which included elimination of clones with incomplete mapping information or missing data in more than 20% of cases. This resulted in 7980 clones evaluable for further analysis. In the next step, genomic events were assigned. The median of all MADs (median absolute deviation) across all chromosomes is taken to estimate the sample experimental variability (genomic "standard deviation", SDg). Gains and losses are defined by log2-ratios larger than 3 times SDg or smaller than - 3 times SDg, respectively.
Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction
After 4 weeks of expansion in 21% and 1% O2, MSC were harvested and total RNA was extracted using peqGOLD TriFast™ reagent (PEQLAB Biotechnologie GmbH, Erlangen) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 1 μg total RNA (Invitrogen, Groningen, The Netherlands) and amplification was done using the following primers: HIF-1α, for: CTCAAAGTCGGACAGCCTCA/rev: CCCTGCAGTAGGTTTCTGCT; GAPDH, for: CGGGAAGCTTGTGATCAATGG/rev: GGCAGTGATGGCATGGACTG. PCR conditions were: HIF-1α, 94°C for 3 min/95°C for 30 s, 56°C for 30 s, 72°C for 1 min (20 cycles)/72°C for 10 min; GAPDH, 95°C for 2 min/95°C for 30 s, 55°C for 30 s, 72°C for 30 s (20 cycles)/72°C for 7 min. Products (358 bp) were separated on 2% agarose gels, visualized with ethidium bromide and analyzed using the gel documentation system AIDA 1D Evaluation (Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany).