CD34+ hematopoietic stem/progenitor cells were isolated from human cord blood and amplified in vitro for 10-14 days in serum-free medium with specific cytokines (Ju et al., Eur. J. Cell Biol. 82, 75-86, 2003; Hacker et al., Nat. Immunol. 4, 380-386, 2003). Cultured progenitor cells were induced to differentiate into DC in RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.1 microM Beta-mercaptoethanol, 100 U/ml penicillin and streptomycin (GIBCO-BRL) and 500 U/ml GM-CSF, 500 U/ml IL-4 for 6 days with or without 10 ng/ml TGF-beta1 as indicated (0.5x10E6 cells/ml). Every 2 days growth factors were added and cells were maintained at 0.5x10E6 cells/ml cell density. DC were left untreated as control.
Treatment protocol
CD34+ hematopoietic stem/progenitor cells were isolated from human cord blood and amplified in vitro for 10-14 days in serum-free medium with specific cytokines (Ju et al., Eur. J. Cell Biol. 82, 75-86, 2003; Hacker et al., Nat. Immunol. 4, 380-386, 2003). Cultured progenitor cells were induced to differentiate into DC in RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.1 microM Beta-mercaptoethanol, 100 U/ml penicillin and streptomycin (GIBCO-BRL) and 500 U/ml GM-CSF, 500 U/ml IL-4 for 6 days with or without 10 ng/ml TGF-beta1 as indicated (0.5x10E6 cells/ml). Every 2 days growth factors were added and cells were maintained at 0.5x10E6 cells/ml cell density. DC were left untreated as control.
Growth protocol
CD34+ hematopoietic stem/progenitor cells were isolated using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity was 83-90%. CD34+ cells (0.3-0.5x10E6 cells/ml) were cultured in StemSpan serum free medium (StemCell Technologies Inc., Vancouver, Canada) with 100 ng/ml SCF, 50 ng/ml Flt3 ligand (FL), 20 ng/ml thrombopoietin (Tpo) and 10 ng/ml hyper-IL-6 (Ju et al., Eur. J. Cell Biol. 82, 75-86, 2003; Hacker et al., Nat. Immunol. 4, 380-386, 2003.). Growth factors were added every 2 days and cells were maintained at 1x10E6 cells/ml cell density. Cells were amplified in vitro for 10-14 days. After 10-14 days of culture progenitor cells were induced to differentiate into DC in RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.1 microM Beta-mercaptoethanol, 100 U/ml penicillin and streptomycin (GIBCO-BRL) and 500 U/ml GM-CSF, 500 U/ml IL-4 for 6 days with or without 10 ng/ml TGF-beta1 as indicated (0.5x10E6 cells/ml). Every 2 days growth factors were added and cells were maintained at 0.5x10E6 cells/ml cell density.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using RNeasy Midi kit (Qiagen, Hilden, Germany) using the manufacturer’s buffers and protocols. An additional DNAse digestion with RNAse free DNAse (Qiagen) was performed. First and second strand cDNA synthesis were done with 7 microg total RNA with the SuperScript Choice system (Gibco BRL) and a T7-(dT)24 Primer (Biotez, Berlin, Germany).
Label
biotinylated UTP and CTP
Label protocol
In vitro transcription employing double stranded cDNA as template and biotinylated UTP and CTP (Perkin Elmer/ENZO) as label was performed with the MEGAscript kit from Ambion according to the manufacturer's instructions. Labeled RNA was fragmented by heating to 95 C for 35 minutes in 200 mM TRIS (pH 8.1), 500 mM KOAc, 150 mM MgOAc buffer.
Hybridization protocol
Affymetrix arrays were prehybridized with 200 microl MES hybridization buffer for 10 minutes at 45 C with rotation (60 U/min) in an Affymetrix hybridization oven 640. After removing the prehybridization solution, 10 microg of labeled cRNA in 200 microl MES hybridization buffer were applied to the array thereby leaving a small air bubble. Arrays were incubated overnight at 45 C with rotation (60 U/min) in the hybridization chamber.
Description
Dendritic cell derived from CD34+ hematopoietic stem/progenitor untreated (control)
Data processing
CEL files were processed with R(www.r-project.org) and Bioconductor (www.bioconductor.org). Scanned array were subjected to multiple quality control tests. This include scaling factor control, control for aberration of average background value, present call percentage on each chip, RNA degradation, images artifact detection.
