CD34+ cells were isolated from fresh umbilical cord blood after written consent according to the guidelines specifically approved by the Ethic Committee of RWTH Aachen University (Permit Number: EK187/08) using the CD34 Micro Bead Kit on a MiniMACS system (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). Cells were cultured in StemSpan serum free expansion medium (Stem Cell Technologies, Grenoble, France) supplemented with 10 µg/mL heparin (ratiopharm, GmbH, Ulm, Germany), 20 ng/mL thrombopoietin (TPO; PeproTech GmbH, Hamburg, Germany), 10 ng/mL stem cell factor (SCF; PeproTech), 10 ng/mL fibroblast growth factor 1 (FGF1; PeproTech). Culture was either performed on tissue culture plastic (TCP) or on a confluent layer of mesenchymal stromal cells (MSCs; passage 3 to 6). After seven days, CD34+ and CD34- fractions were again separated as described above.
Extracted molecule
total RNA
Extraction protocol
RNeasy kit (Qiagen)
Label
biotin
Label protocol
biotin
Hybridization protocol
See Affymetrix protocols
Scan protocol
Arrays were scanned using Affymetrix GeneChip Scanner 3000.
Data processing
CEL files were processed using the Affymetrix Power Tools (APT) Software Package. Normalization was done employing RMA (Irizarry et al., Biostatistics 4, 249-264, 2003).
