Mesenchymal Stem Cells of Patients with Chronic Myeloid Leukemia Are Philadelphia Negative.

Abstract

In the last years, focus of regenerational studies has pointed on mesenchymal stem cells (MSC) and their ability to differentiate into several mesenchymal tissues. MSC have been shown to play a pivotal role in the microenvironment of bone marrow cells and in the modulation of immune response as they can suppress lymphocytic proliferation in vitro. Moreover, some animal studies have suggested they could favor the proliferation of malignant cell clones in solid tumor models. Their role in hematological malignancies, however, remains to be further elucidated and little is known about the influence of MSC in the development and maintenance of the malignant clone in chronic myeloid leukemia (CML). This disease is characterized by the presence of the Philadelphia (Ph) chromosome, a fusion product generated by the reciprocal translocation between chromosomes 9 and 22. Previous reports showed that hepatocytes precursors, found in the liver of CML patients carry the Ph translocation. Our intent was to elucidate whether MSC isolated from patients with CML in different stages of the disease originate from the malignant clone. To this purpose bone marrow aspirates of 11 patients with CML were obtained after informed consent. Five patients were analyzed at diagnosis, two after allogenic stem cell transplantation, three on treatment with the tyrosine kinase inhibitor imatinib and one on treatment with interferon alpha in combination with hydroxyurea. MSC were then generated as previously described. Briefly, cells were isolated by density gradient methods, resuspended in RPMI1640 medium containing 10% fetal bovine serum and plated in culture flasks to adhere. After 4–5 weeks of culture cells were collected and characterized by the expression of several surface markers in a fluorescence activated cell sorter (FACS). The presence of the Ph chromosome was assessed by both fluorescence in situ hybridization (FISH) and polymerase chain reaction (nested PCR). Moreover whole bone marrow was analyzed and results compared with those obtained in the MSC population. MSC showed a typical morphological pattern, growing to confluence after a few weeks of culture and appearing as an adherent, spindle shaped cell layer. In FACS they stained positive for SH2 and SH3 and did not express CD34, CD45 and CD14. MSC were then analyzed by FISH using probes for BCR-ABL. We could not detect the Ph translocation in any of the analyzed patients, though it was present at variuos levels in the remnant bone marrow cells. Results did not change, if expression of BCR-ABL was measured by high sensitivity RT-PCR. Our results showh that MSC of patients with CML are Philadelphia negative irrespective of the stage of disease and the treatment given, suggesting that these cells are not involved in the development of the malignancy. However, their interactions with leukemic cells as well as their role in the immune response against the tumor remains to be further characterized.