ABSTRACT: Myelodysplastic syndromes (MDS) are a group of clonal disorders of hematopoietic stem cells. Mesenchymal stem cells (MSC) are the progenitors of the Bone Marrow (BM) stroma and have been involved in the physiopathology of MDS. The presence of cytogenetic aberrations on MSC from MDS patients is controversial. The aim of the study is to characterize BM derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridisation (FISH) analysis and genetic changes by array based comparative genomic hybridization (array-CGH). 32 cases of untreated MDS were included in the study. MSC from MDS achieved confluence at a slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was also lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic, and gains were more frequent than looses. The results of array-CGH were confirmed by FISH. When an unsupervised hierarchical cluster analysis was performed two clusters were identified: one of them included the 5q- syndrome patients, while the other incorporated the rest of the MDS patients. Our results shows, for the first time, that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to particular MDS subtypes. Keywords: Genomic comparison between mesenchymal cells 32 BM cells from âde novoâ MDS, corresponding to 29 patients, were included in the study. Male/female ratio was 14/15 and median age was 70 years (range 34-89). MDS diagnosis was established by peripheral blood counts and morphology, BM morphology and cytogenetic analyses and classified according to the WHO proposals. 12 Patientsâ characteristics are summarized in table 1. Three patients were included twice in the study: at the diagnosis of one RA (case 2) and two Unclassifiable MDS (cases 29,30), and at the progression into RAEB-2 (cases 16, 20 and 18). None of them had received treatment during this period of time, other than supportive therapy. Normal BM samples were obtained from 15 healthy donors, 8 males and 7 females, with a median age of 57 (range 37-88). Written informed consent was obtained from all patients and donors according to ethical guidelines of our institution. Genomic-wide analysis of DNA copy number changes of MSC from 13 cases was performed using array-CGH. Slides containing 3032 BACs were produced in âCentro de Investigación del Cáncerâ (Salamanca, Spain). The particular BAC and P-1 derived artificial chromosome set used to produce this array is distributed to academic institutions by the Welcome Trust Sanger Institute (Cambridge, United Kingdom) and contains targets spaced at ï» 1 Mb density over full genome, a set of subtelomeric sequences for each chromosome arm, and a few hundred probes selected for their involvement in oncogenesis. The clone content is available in the âCytoviewâ windows of the Sanger Institute mapping database site, Ensembl (http://www.ensembl.org/). According to this database, clones were ordered along the chromosomes. These clones were isolated from their bacterial cultures with the relevant antibiotics and the DNA was extracted with standard protocol Welcome Trust Sanger Institute (Cambridge, UK). 10 ng of DNA (BAC/PAC) was used as a template for three DOP-PC. 22;23 These products were ethanol precipitated and dissolved in distilled water. A minimum of three replicates per clone were printed by Microgrid II (Biorobotics) on each slide (Ultragaps Coated Slides, Corning) using the aqueous dimethylsulfoxide buffer as spotting solution. Briefly, for labelling reactions, 1 μg of nonamplified genomic DNA, test and reference were digested separately with DpnII restriction enzyme (New England Biolabs, Beverly, MA). For microarray hybridization, the digested DNAs were separately labelled using random primers (Bioprimer labelling kit, Invitrogen) and cyanine dye-3-dCTP and cyanine dye-5-dCTP fluorescent dye to paired hybridisation samples (Amersham Biosciences). The incorporated of the label nucleotide was quantified using the Nanodrop spectrophotometer. Labelled test and reference DNAs were mixed equitably, co-precipitated in presence of Cot-1 human DNA (Roche, Indianapolis, IN) with ethanol, washed, and resuspended in hybridization solution (50% Formamide, 10% Dextran sulfate, 2X standard saline citrate, 10 mM Tris pH 7.6, 2.7% sodium dodecyl sulfate and 10 μg/ μl of yeast tRNA). DNA mixtures were hybridized to the arrays in the TECAN HS4800 Pro according to the manufacturerâs recommended protocol. Images and signal intensities were acquired using GenePix4000B (Axon Instruments, Burlingame, CA) dual laser scanner in combination with GenePixPro4.0 (Axon Instruments) imaging software. Image and Data analysis. Spot intensities measured by the GenePix Pro 4.1 software (Axon Instruments, Wetsbur BV, Leuden, The Netherland). Pixel intensities for each feature were integrated and median values were determined, and the local background was calculated. For each spot the intensities were corrected by subtracting the local background for both wavelengths, and only spots with signal intensities al least threefold above background signal intensities were included in the analysis. The median of the ratios of all spots was calculated and used to normalize all data points. From each feature, the log2 value of the average of the normalization ratios of the spots was calculated and used to display the data. In addition, fluorescence ratios were normalized for each microarray using the print-tip loess method and the background subtraction with the Diagnosis and Normalization Arrays Data tool. 24 The cut-off level was determined for each individual experiment after avering the ratios from the two color-swith hybridizations and subsequent for normalization, and calculate the mean and Standard derivations to define the cut-off level as means plus/minus two times the standard derivations. The clones that showed possible changes were submitted to the Database of Genomics Variants (http://projects.tcag.ca/variation/) and chromosomal Imbalances and Phenotype in Humans using Ensembl Resources (DECIPCHER: http: www sanger. ac.uk /PostGenomics/decipher/. Clustering Unsupervised clustering We converted the relative ratio value for each BAC clones in each sample to score of 1 (gain/amplified), 0 (no change), or -1 (loss) based on the 2 SD standards described above and analyzed with Cluster and TreeView) 25 (Cluster and TreeView software) based on the average linkage method with the centered correlation metric was used (or Pearson Uncentered). Supervised Sam analysis was used http://www-stat.stanford.edu/ tibs/SAM) 26 to identify BACs that segregate subgroups on MSC. BACs with an SD of zero across the data set before analysis were removed.
