tissue: Bone Marrow Mesenchymal Stem Cells time: 3 weeks treatment: treated with 1 mM ATP
Treatment protocol
BM-hMSCs were treated with 1 mM ATP or with solvent alone 24 hours before to initiate osteogenic or adipogenic differentiation protocols. To induce adipogenic differentiation BM-hMSC cells were seeded at 2.1 x 104 cells/cm2 on Lab-Tek II coverglass chamber (Nalge-Nunc, Roskilde, Denmark), were grown for 3 days in Adipogenic induction medium (Lonza) containing additional h-Insulin, L-glutamine, MCGS, Dexamethasone, Indomethacin, 3-isobuty-I-methyl-xanthine, Pen/Strep followed by 3 days in Adipogenic maintenance medium containing h-Insulin, L-glutamine, MCGS, Pen/Strep. Both cycles were repeated and cell cultures were stopped at day 12 and 18, for histological staining and total RNA extraction. To induce osteogenic differentiation BM-hMSC cells were seeded at 3.1 x 103 cells/cm2, were grown in Osteogenic Differentiation medium (Lonza) containing L-glutamine, MCGS, Dexamethasone, Ascorbate, β-Glycerophosphate, Pen/Strep. The media was replaced every 3-4 days. Cell cultures were stopped at day 14 and 21 for histological staining and total RNA extraction.
Growth protocol
hMSCs were isolated from BM aspirates of normal donors after obtaining informed consent, as previously described (Aluigi et al., 2006). Briefly, the mononuclear cell fraction was separated by centrifugation over a Ficoll-Paque gradient (Amersham Bioscience, Piscataway, NJ, USA), resuspended in proliferation medium consisting of low-glucose Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Milan, Italy), 10% bovine serum albumine (BSA) (Gibco-Invitrogen, Carlsbad, CA, USA), 2 mM L-glutamine, and 1% antibiotics (MP Biomedicals, Verona, Italy), and plated at an initial seeding density of 1.6x105 cells/cm2. After 3 days, the non-adherent cell fraction was removed by washing with phosphate-buffered saline (PBS), and monolayers of adherent cells were cultured until they reached 70%–90% confluence. Cells were then trypsinized (0.25% trypsin with 0.1% EDTA) (EuroClone, Pero, Italy), subcultured at a density of 3.5 x 103 cells/cm2, and used for further experiments within passages 3 to 5.
Extracted molecule
total RNA
Extraction protocol
ATP pre-treated or untreated adipocyte, osteoblast and BM-hMSCs were collected and lyzed in QIAZOL Buffer (Qiagen, Valencia, CA, USA). Total RNA from BM-hMSCs was extracted using miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s recommendations. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit; Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration and purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to evaluate the RNA sample concentration, and 260/280 and 260/230 nm ratios were considered to verify the purity of total RNA.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard GeneAtlas 3’IVT Express Kit protocol from 100ng of total RNA (P/N 702833 Rev. 4, Affymetrix). Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the cRNA concentration/quality as well as to optimize the cRNA fragmentation.
Hybridization protocol
Fragmented cRNA was hybridized for 16 hr at 45C on Affymetrix HG-U219 array strips in GeneAtlas Hybridization Station. GeneChips were washed and stained using GeneAtlas Hybridization, Wash, and Stain Kit for 3’IVT Arrays.
Scan protocol
GeneChip were finally scanned using GeneAtlas Scanner.
Description
Mesenchymal Stem Cells (MSC) pre-treated with 1mM ATP
Data processing
Gene expression data were imported into Partek Genomics Suite 6.5 (Partek, St Louis, Mo) as CEL files using default parameters. Raw data were preprocessed, including background correction, normalization, and summarization using robust multiarray average analysis.
