Cells were harvested with 0.05% Trypsin+EDTA, washed with PBS and stored in RNAlater at -80˚C until RNA was extracted
Growth protocol
Human MAPC were generated by flushing the bone fragment and plating the total cell fraction at 0.5x106 cells/cm2 in medium consisting of 60% DMEM low-glucose (Gibco, Invitrogen, Carlsbad, CA, USA), 40% MCDB-201 (Sigma-Aldrich, St Louis, MO, USA), supplemented with 50nM dexamethasone, 10-4M L-Ascorbic Acid, 1xSelenium-Insulin-Transferrin (ITS), 0.5xlinoleic acid-bovine serum albumin (LA-BSA) (all from Sigma-Aldrich), 1% Penicillin/Streptomycin (Gibco, Invitrogen), along with 2% Serum Supreme (Lonza BioWhittaker, Basel, Switzerland) and human PDGF-BB (R&D Systems, Minneapolis, MN, USA) and human EGF (Sigma-Aldrich) (both 10ng/ml). Human MAPC cultures were maintained under hypoxic conditions (5%O2) in a 5.5% CO2 humidified incubator at a density of 400 cells/cm2 and were passaged every 2-3 days. Clonal populations were obtained by plating 5 cells/well in a 96-well or 48-well plate between passage 2-12. Human MSC were generated by flushing the bone fragment and plating the mononuclear fraction, obtained after Ficoll density gradient centrifugation, at 0.5x106 cells/cm2 in MSC growth medium (Lonza). Human MSC cultures were maintained at 5,000 cells/cm2, at normal oxygen levels in a 5% CO2 humidified incubator and were passaged every 4-7 days. To isolate hMab, the muscle fragment was rinsed in PBS (w/o Ca2+Mg2+), cut into very small pieces (1-2 mm diameter) and transferred to a Petri dish coated with type I collagen (Sigma-Aldrich). The medium consisted of MegaCell DMEM (Sigma-Aldrich) supplemented with 5% FBS (Lonza BioWhittaker), 5ng/ml bFGF (R&D Systems), 2mM L-glutamine, 0.1mM β–mercaptoethanol, 1% non-essential aminoacids (NEAA) and 1% Penicillin/Streptomycin (all from Gibco, Invitrogen). The tissue fragments were cultured for 7-10 days and after the initial outgrowth of fibroblast-like cells, small round and refractile cells could be observed. Human Mab cultures were maintained at 5,000 cells/cm2, under hypoxic conditions (5%O2) in a 5.5% CO2 humidified incubator and were split every 2-3 days. The human Embryonic Stem Cell (hESC) line H9 (purchased from WiCell, Madison, WI, USA) was obtained with approval from the Ethical Committee of the K.U.Leuven. Cells were cultured as described on mitomycin-inactivated mouse embryonic fibroblasts (MEFs) in hESC expansion medium in a 5% CO2 normoxic humidified incubator and passaged 1:3 using collagenase IV (Gibco, Invitrogen) every 3-7 days.46 To obtain samples for RNA extraction, cells were cultured feeder-free in mTeSR medium (Stem Cell Technologies, Vancouver, Canada) on Matrigel (hESC-qualified Matrix, BD Biosciences, San Jose, CA, USA). Human MSC were obtained from Lonza and cultured under the same conditions as the other MSC.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated using standard RNA extraction protocols (Trizol) and RNA quality was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 1 ug total RNA using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. Dye incorporation rate and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 1.65µg Cy3-labeled cRNA was hybridized overnight (17hrs, 65˚C) to Agilent Whole Human Genome Microarrays 4x44K using Agilent's recommended hybridization chamber and oven. After hybridization, microarrays were washed once with 6xSSPE buffer containing 0.005% N-lauroylsarcosine for 1min at room temperature followed by a second wash with preheated 0.06xSSPE buffer containing 0.005% N-lauroylsarcosine for 1min. The last washing step was performed with acetonitrile for 30sec
Scan protocol
Slides were scanned using Agilent Technologies Scanner G2505C US22502695 with one channel protocol, 5 um scan resolution
Description
RNA extracted from cells cultured under MAPC conditions
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using protocol GE1-v5_10_Apr08 and Grid: 014850_D_F_2007082 to obtain Processed Signal intensities. The signal intensities (gProcessedSignal) for each array were normalized by dividing the probe intensities by the array median signal intensity (median calculated after excluding control probes). The resulting values were log2 transformed to yield median normalized signal intensities. Probes whose signal intensity was not positive and significant above the background as determined by FES (gIsPosAndSignif) in at least two of the samples being compared were filtered out from analysis. Probe summarization (exclusion of duplicated probes) was performed in R Bioconductor environment using the Agi4x44PreProcess package.
