Abstract
Bone marrow-derived mesenchymal stem cells (BM-MSC) have the capacity to differentiate into various cell types in specific conditions, to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. However, little is known about the molecular genetics and cytogenetics of these cells. In this study, we established and expanded BM-MSC from bone marrow of patients with acute myeloid leukemias (AML). A total of 10 cases including from patients with normal (n=7) and abnormal karyotypes (n=3) were analyzed by conventional cytogenetic methods, array-CGH and FISH. Ten metaphases were analyzed in each case. Genomic DNA from the MSC was extracted and array comparative genomic hybridization (a CGH) was performed using the PerkinElmer Constitutional Chip 4.0 that contains 5,200 BAC clones printed in triplicate that detects and maps changes in copy number of DNA sequences. In a CGH, DNA from MSC and a reference genome (genomic DNA from a normal individual) are differentially labeled with fluorescent dyes and hybridized to a representation of the human genome, in this assay a DNA microarray containing BAC clones with human DNA inserts. Hybridization of repetitive sequences is blocked by the addition of Cot-1 DNA. The fluorescence ratio of the test and reference hybridization signals is determined at different positions along the genome and provides information on the relative copy number of sequences in the test genome compared with a normal diploid genome. The final plots were generated by SpectralWare software using appropriate normalization. For confirmation, individual BAC DNAs were labeled using the Invitrogen DNA labeling kit for FISH. Results demonstrate that G-banding analysis did not show chromosome rearrangement in 9 cases. There was 47,XX, +5 in one case that had 46, XX, der(16)t(1;16)(q21;q12.1) in the primary AML sample. This finding was confirmed by FISH and aCGH. At variance to BM-MSC derived from normal donors, all 10 cases of AML-derived MSC showed abnormalities (gain or loss) in different regions of chromosomes. The most frequently involved chromosomes were No. 3, 4, 6, 7, 8, 10, 15, 16, 19, and 22. The abnormalities were confirmed by FISH.
Case CG of primary samples CG in MSC aCGH-results 1 46,XY 46, XY Gain-16q11.2q12.1) Gain-3p26.3 Gain-14q31.3 2 46XX,del(5)(q13q33) 46, XX Loss-7p21.3 Gain-3p26.3b Gain-16p12 3 46XX, der(16)t(1;16) (q21;q12.1) 46, XX(9), 47,XX,+5(1) Gain-5 4 46, XY 46, XY Gain-10q11.22 Gain-7q11.23 Loss-17q12 5 46,XX 46, XX Loss-15p13 Loss-3q12.3 6 46,XY, 46XY,del(5)(p13q13)(2) 46, XY Gain-10qter Gain-2q37.3 Loss-3p26.3 Gain-15q11.2 7 46, XY 46, XY Gain-5q14 8 46, XY 46, XY Gain-4q35.2 Gain-6q16 Loss-15q11.2 loss-16p11.2 Loss-22q12 9 46, XY 46, XY loss-8q21.3q22 Loss-7p21.3 10 46, XY 46, XY loss-19p13.2 Loss-8q21.2b Loss-14q12
These results indicate that BM-MSC derived from AML have genomic abnormalities which may be important for their support of leukemia cell growth and resistance to chemotherapeutic agents.
437 DETERMINING GENE COPY NUMBER IN TRANSFECTED CAPRINE FIBROBLAST CELLS