Mesenchymal Stem Cells from Patients with Myelodysplastic Syndromes Are Functionally and Genomically Abnormal.

Abstract

Myelodysplastic syndromes (MDS) are a group of clonal disorders of hematopoietic stem cell (HSC). The hematopoietic microenvironment plays a major role in the physiology of the hematopoietic system, and mesenchymal stem cells (MSC) are not only the progenitors but also the key regulators of this microenviroment. Recently, some data has been published showing that these MSC could be involved in the MDS pathophysiology. Moreover, the presence of cytogenetic aberrations on these cells is controversial. The aim of the study was to characterize bone marrow derived MSC from patients with MDS using different approaches: kinetic studies, immunophenotypic analysis and genetic changes by array-based comparative genomic hybridization (array-CGH). FISH was performed with the probe of 1q31 and Q-PCR was performed with the SYBR Green technique in order to confirm array-CGH results. Patients with untreated MDS were included in the study. Median age was 72 years (range: 54–89). Diagnosis of MDS was established according to the WHO classification as follows: 5q- syndrome (n=7), refractory anemia (n=2), refractory anemia with ringed sideroblasts (n=1) and refractory anemia with excess blasts type 2 (n=3). Standard cytogenetic and FISH studies on hematopoietic cells were performed at diagnosis according to standard methods. MSC from MDS were compared to those from 12 healthy donors. MSC were obtained by plating mononuclear cells from bone marrow, and cultured and expanded following standard procedures. According to the international consensus for MSC characterization, in the third passage MSC were harvested to perform phenotypical studies and cytogenetics. To perform Array-CGH a total of 3500 genomic targets were compounded from RP-11 libraries. The PCR products after purification were arrayed onto glass slides using a BioRobot. DNA was labelled, denaturalised and hybridizated. MSC from MDS achieved confluence at a slower rate than donor-MSC [23 days (range 12–90) vs 15 days (range 11–30) p<0,01]. Also some phenotypical markers showed lower expression on patients MSC: CD105 and CD104 (p<0,05%), compared to MSC from bone marrow donors. In all MDS cases analysed MSC showed DNA genomic changes. The most frequent aberrations were 1q31q32 region gains, present in 72% of cases, and 5q31 loss in 46% of patients. Gains in 1q31 were confirmed by FISH using the probe obtained from the BAC. Loss of 17p13 occurred in 3 cases (28%), and this may be relevant since p53 is included in that region, Q-PCR was subsequently performed confirming the loss of p53 in all these cases. The changes were not observed in hematopoietic cells analysed in order to exclude somatic changes. We conclude that MSC from MDS are functionally abnormal since they show a slower growing capacity and a lower expression of adhesion molecules. In the present study it is shown for the first time that MSC from MDS show several genomic aberrations when CGH arrays are used and the data have been confirmed by FISH and Q-PCR.