Leukemia (2009) 23, 1515–1527 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE Mesenchymal stem cells from multiple myeloma patients display distinct genomic profile as compared with those from normal donors M Garayoa1,5,6, JL Garcia1,2,6, C Santamaria3, A Garcia-Gomez1, JF Blanco4, A Pandiella1, JM Herna´ndez3, FM Sanchez-Guijo3,5, M-C del Can˜izo3,5, NC Gutie´rrez3, and JF San Miguel1,3,5 1Centro de Investigacio´n del Ca´ncer, Instituto de Biologı´a Molecular y Celular del Ca´ncer, Universidad de Salamanca-CSIC, Salamanca, Spain; 2Unidad de Investigacio´n, Instituto de Estudio de Ciencias de la Salud de Castilla y Leo´n (IECSCYL) – Hospital Universitario de Salamanca. Salamanca, Spain; 3Servicio de Hematologı´a. Hospital Universitario de Salamanca. Salamanca, Spain; 4Servicio de Traumatologı´a. Hospital Universitario de Salamanca. Salamanca, Spain and 5Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leo´n, Salamanca, Spain It is an open question whether in multiple myeloma (MM) bone directly by interactions of myeloma cells with BM stromal cells marrow stromal cells contain genomic alterations, which may and extracellular matrix proteins or indirectly by secretion of contribute to the pathogenesis of the disease. We conducted an array-based comparative genomic hybridization (array-CGH) soluble cytokine and growth factors by myeloma cells and/or analysis to compare the extent of unbalanced genomic altera- stromal cells. These interactions and growth factor circuits tions in mesenchymal stem cells from 21 myeloma patients ultimately lead to the activation of pleiotrophic signalling (MM-MSCs) and 12 normal donors (ND-MSCs) after in vitro cascades, which promote proliferation, cell survival, anti- culture expansion. Whereas ND-MSCs were devoid of genomic apoptotic signalling, drug resistance and migration of MM imbalances, several non-recurrent chromosomal gains and cells.4,5,7 Furthermore, crosstalk of myeloma cells with other losses (41 Mb size) were detected in MM-MSCs. Using real- time reverse transcription PCR, we found correlative deregu- cells in the BM milieu, such as osteoblasts and osteoclasts, lated expression for five genes encoded in regions for which results in inhibition of the former and activation of the latter, genomic imbalances were detected using array-CGH. In addi- which produces an unbalanced bone remodelling responsible tion, only MM-MSCs showed a specific pattern of ‘hot-spot’ for the development of bone lesions in MM patients.6,8,9 regions with discrete (o1 Mb) genomic alterations, some of Bone marrow mesenchymal stem cells (MSCs) are an essential which were confirmed using fluorescence in situ hybridization cell type in the formation and function of the BM micro- (FISH). Within MM-MSC samples, unsupervised cluster analysis did not correlate with particular clinicobiological features of MM environment, being the progenitor cells of osteoblasts and patients. We also explored whether cytogenetic abnormalities the haemopoietic-supporting stroma components of the 10,11 present in myelomatous plasma cells (PCs) were shared by marrow. In fact, differences between MSCs derived from matching MSCs from the same patients using FISH. All MM- MM patients (MM-MSCs) and those from healthy donors MSCs were cytogenetically normal for the tested genomic (ND-MSCs) have been reported.12–16 When compared with alterations. Therefore we cannot support a common progenitor their normal counterparts, MM-MSCs differ in spontaneous and for myeloma PCs and MSCs. Leukemia (2009) 23, 1515–1527; doi:10.1038/leu.2009.65; myeloma cell-induced production of cytokines, exhibit a published online 9 April 2009 decreased proliferative capacity and present reduced efficiency Keywords: array-CGH; mesenchymal stem cells; multiple to inhibit T-cell proliferation, and the osteoblasts derived from myeloma; genetic alteration MM-MSCs show a diminished matrix mineralization potential when compared with their normal counterparts.12–16 Moreover, Corre et al.13 have observed a distinctive gene expression profile for MM-MSCs and ND-MSCs upon using microarray analysis, with differential expression of genes coding for growth and Introduction angiogenic factors, as well as for factors related to bone differentiation.13 All these differences were detected after MSCs Multiple myeloma (MM) is a B-cell neoplasia characterized by isolation and expansion in culture. Although it has been accumulation of clonal plasma cells (PC) in the bone marrow, suggested that these differences between normal and myeloma- the presence of monoclonal immunoglobulin in blood and/or tous MSCs could be attributed to the presence of genomic urine, and the existence of bone lesions. The genetic basis of the alterations in MM-MSCs,12–14 this is still an issue to be clarified. disease includes recurrent and complex genetic abnormalities in On the other hand, the clinical observation that bone lesions myelomatous cells, which besides contributing to the pathogen- from MM patients do not heal even after response to therapy9,17 esis of MM also influences the heterogeneous clinical course of also seems to support the idea of a permanent defect in the 1–3 the disease. In addition, the involvement of the bone marrow capacity of MM-MSCs to generate fully functional bone- (BM) microenvironment in the pathophysiology of the disease is generating osteoblasts. This last issue could also be explained 4–7 nowadays well-accepted. This contribution is mediated either by genomic alterations present in MM-MSCs, which would remain in the absence of myeloma cells. Correspondence: Dr M Garayoa, Centro de Investigacio´n del Ca´ncer - An additional controversial issue in the field of MSCs is the CSIC, Campus Miguel de Unamuno, Avda. Coimbra s/n. 37007, idea of a common haematopoietic and mesenchymal progeni- Salamanca, Spain. tor, with occasional reports favouring this possibility.18–20 E-mail: [email protected] 6These two authors contributed equally to this work. Theoretically, if MSCs from myeloma patients would encompass Received 30 September 2008; revised 25 February 2009; accepted 27 cytogenetic markers present in myelomatous PCs, the genomic February 2009; published online 9 April 2009 events leading to those genomic aberrations should have Genomic profile of multiple myeloma MSCs M Garayoa et al 1516 occurred earlier to a mesenchymal or haematopoietic commit- Healthcare, Uppsala, Sweden) and cultured in low-glucose ment, thus supporting the idea of a common precursor for both Dubecco’s modified Eagle’s medium supplemented with 10% types of cells. heat-inactivated fetal bovine serum, 100 U/ml penicillin, To gain insight into these questions, we carried out a genome- 100 mg/ml streptomycin and 1% L-glutamine. After 3 or 4 days wide scan on MSCs derived from MM patients and healthy in culture, non-adherent cells were removed, whereas MSCs donors using array-based comparative genomic hybridization were selected by their adherence to the plasticware (this was (array-CGH). This technique has already been successfully used considered as passage 0, P0).10 For five selected donors, the to explore the presence of gains and losses of genetic material in non-adherent fraction was not discarded, but harvested to several types of cancer21–25 and also to discriminate, within the obtain a normal control DNA for array-CGH hybridization. The genomic alteration, the contribution of the stromal vs epithelial culture medium was replaced twice weekly until MSC cultures compartments.26 Besides, we have also explored the presence of were approximately 90% confluent or had remained a maxi- genomic alterations of myelomatous PCs in the matching MSCs mum of 21 days in culture; at this point, cells were trypsinized from the same patients using fluorescence in situ hybridization (0.05% trypsin-EDTA) and expanded in a 1:3 ratio (P1). The (FISH). MSCs were collected near confluency at P3 for subsequent genomic DNA isolation or FISH studies. No apparent haema- tological cell contamination was observed under the micro- Materials and methods scope at the time of MSC harvest. The cultures were maintained at 37 1C and 5% CO2. All the cell culture media and reagents Participants were purchased from Invitrogen (Paisley, UK). A total of 26 patients with newly diagnosed MM were included Selected MSCs from both MM patients (n ¼ 4) and healthy in this study (median age was 68 years, ranging from 28 to 89 donors (n ¼ 4) at P3 were also tested to meet the minimal criteria years). Characteristics of the patients are listed in Table 1. A total as defined by the International Society for Cellular Therapy for of 12 healthy controls of BM samples were obtained from multipotent mesenchymal stromal cells.27 These criteria in- participants undergoing orthopaedic surgery, with a similar age cluded specific cell surface antigen expression and trilineage range to MM patients (median age 58 years, range 26–88 years). mesenchymal differentiation potential. Combinations of mono- Every sample was obtained after receiving informed written clonal antibodies anti-CD34-APC (allophycocyanine), -CD19- consent of patients and donor volunteers and following approval APC, -CD45-PerCPCy5.5, -HLA-DR-PerCPCyC5.5, -CD14-FITC from the Ethical Committee of our Institution. (fluorescein isothiocyanate) (Becton-Dickinson Biosciences, San Jose´, CA, USA), -CD90-FITC (allophycocyanine), -CD73- PE, -CD166-PE, -CD106-PE (BD Biosciences Pharmingen, San MSC harvest, culture conditions and characterization