Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that contribute to the structure and function of the bone marrow (BM) niche, controlling homing, self-renewal, differentiation and proliferation of hematopoietic stem/progenitor cells (HSPCs).Recently, genetic and functional alterations of MSCs have been reported to occur in patients affected by myelodysplastic syndromes (MDS) and acute leukemias in keeping with the exptl. notion that MSC dysfunction can affect relevant functions of leukemic blasts and induce MDS and leukemia in murine models.5 Philadelphia-neg. myeloproliferative disorders (Ph-neg MPNs) encompass polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and post-PV or -ET myelofibrosis.Very little is known about BM-derived MSCs of patients with MPNs apart from the absence of the V617FJAK2 mutation,6 the mol. marker of hematopoietic cells in a great proportion of MPN patients.We have functionally and genetically characterized MSCs from 38 Ph-neg MPN patients and 15 healthy donors (HDs) (Figure 1a) for clin. and epidemiol. characteristics.The study was approved by the institutional review board of IRCSS Policlinico San Matteo Foundation; patients and HDs gave written informed consent.MSCs were isolated either from BM aspirates or/and from trephine bone fragments (TBF) digested with collagenase, expanded and immunophenotypically characterized as described.No significant differences were observed between BM or TBF-expanded MSCs (not shown), as already reported.Both patient and HD MSCs displayed similar morphol., and expressed comparable high levels of CD90, CD73, CD105, CD13 and HLA A-B-C surface antigens, whereas they were neg. for CD33, CD34, CD45, CD14, CD80, CD31 and HLA-DR mols.The median expression of CD146 was lower (32%, range 5-77) in MSCs from MPN patients than that from HDs (44%, range 27-80), although the difference did not reach statistical significance (P=0.2).On the contrary, the median percentage of nestin-expressing MSCs was higher in patients than in HDs (72%, range 31-97 vs 54.5%, range 24-82, resp.; P=0.041; Figure 1b).Patient MSCs also displayed higher nestin mean fluorescence intensity than HDs (Figure 1b; P=0.035).Despite similar clonogenic efficiency, the population-doubling time of MPN MSCs was higher than that of HDs; in particular, patient MSCs showed slower proliferation rate at P1-P2 than HD MSCs (P=0.032), with the lowest rate shown by MSCs from PMF patients (Figure 1c).The latter also reached senescence at earlier passages compared with HD MSCs (P=0.015; Figure 1d).Osteoblastic and adipogenic differentiation capacity was observed both in patient and in HD MSCs; however, patient MSCs showed lower osteogenic differentiation capacity (assessed as calcium deposition, measured spectrophotometrically at 550 nm after extraction of Alizarin red) compared with their HD counterpart (median value: 70, range 14-515 vs 319 μg/mL, 53-1940, resp., P=0.003).To analyze the capacity of patient MSCs to support long-term hematopoiesis in vitro, HD-derived CD34+ cells were co-cultured on feeder layers of irradiated early-passage MSCs, as previously reported.Patient MSCs supported long-term hematopoiesis at a significantly lower extent (P=0.036) than those from HDs (median yield of hematopoietic colony-forming unit (CFU): 3, range 0-12 vs 7, range 2-13, resp.).Cultures with MSCs carrying cytogenetic abnormalities (see below, n=4) showed a lower yield of hematopoietic CFU (median 1, range 0-3) than those without genetic abnormalities (n=9, median 3, range 1-12), the difference, however, not reaching a statistical difference (P=0.08).DNA was extracted from MSCs of both patients and HDs at different passages.Early-passage MSCs of 20 patients with PMF and 8 with PV/ET, known to carry the JAK2V617F mutation in their granulocytes, never showed the mutation at PCR anal., as previously reported.6 Array CGH was performed in MSCs of 23 out of 28 PMF patients, in 6 out of 10 PV/ET patients and in all HDs.Genetic abnormalities were found in 4 PMF patients and in 1 PV patient, whereas HD MSCs never showed genetic abnormalities.As shown in Table 1, genetic alterations included both loss and gain of material, and affected different chromosomes (see Figure 1e for a representative array CGH profile).In some cases, they were detectable at very early (P0-P2) or intermediate passages (P5-P7), whereas in others they appeared later (P11-P13).Array CGH performed on peripheral blood granulocytes never showed the corresponding genetic abnormalities; conversely, one patient without genetic abnormalities in MSCs harbored a chromosome 1q deletion in his granulocytes.The presence of genetic abnormalities was confirmed by reverse transcription PCR on DNA of MSCs at the same passage at which the abnormalities were detected by array CGH (not shown).Patients with genetically aberrant MSCs had higher white blood cell count (P=0.02) and spleen index (P=0.05), calculated as the product of the length of the longitudinal axis by that of the transverse axis (defined as the maximal width of the organ), compared with patients without.Demog., disease duration, time of MSC senescence, Hb level, platelet count and mutational JAK2 status were similar in patients with and without genetic abnormalities.In nine female patients (five PMF, one PV and three ET), polymorphic at the HUMARA locus, the pattern of X-chromosome inactivation (XCI) was studied by means of HUMARA assay, as described.A clonal population of MSCs was found in one PMF, one PV and one ET patient (mean age 41±7 years), both at early and late passages.CFU fibroblast frequencies in these patients were 12, 14 and 9 colonies × 106 mononuclear cells plated, and none of them had chromosomal abnormalities at array CGH.Five informative HD females (mean age 38±6 years) showed a polyclonal pattern of XCI.Our results clearly indicate that MSCs from MPN patients display functional and genetic abnormalities compared with their HD-derived counterparts.A major functional defect of MSCs in patients with MPNs is their low proliferative potential, together with precocious senescence and reduced osteogenic differentiation potential.The functional defects of MPN-derived MSCs are similar to those reported by Gehy et al.4 in MDS-derived MSCs, which also showed an abnormal expression of Jagged-2, osteopontin, c-kit ligand and angiopoietin.The altered expression of these mols. can be responsible for defective cross-talk between MSCs and HSPCs in MDS patients.We did not evaluate the expression of these factors; however, we observed an increased levels of nestin in MSCs of Ph-neg MPN patients that could cause per se a defective HSPC support, in keeping with results of a recently published mouse model, in which a population of nestin-neg. and CXCL12-pos.MSCs was demonstrated to be required for HSPC maintenance.These observations, together with the reduced capacity of sustaining long-term hematopoiesis in vitro, suggest a deep perturbation of the hematopoietic niche in MPN patients and could account for the increased trafficking of HSPCs that characterizes patients with PMF.These results are further supported by the demonstration that in human BM sections from both HDs and MDS patients, most HSPCs are in close contact with CD271+ nestin-neg. MSCs.In agreement with previous studies in MDS and acute leukemia,2, 3, 4 we observed that 17% of patients with Ph-neg MPNs developed heterogeneous genetic abnormalities that were often complex, affected different chromosomes, appeared both at early and late passages, did not confer a proliferative advantage and were not detected in the hematopoietic cells of the patients.Extensive in vitro culture of MSCs could account for the appearance of genetic abnormalities due to the replicative stress; however, employing a standardized culture method implemented in cooperation with the European Group for Blood and Bone Marrow Transplantation Mesenchymal Stem Cell Consortium, we never detected genetic abnormalities in several MSC lots obtained from HD BM, even at very late passages (P>15).Moreover, the detection of an elevated number of mutated cells in one PMF patient after only one passage in vitro supports the concept of a high predisposition of MPN-derived MSCs to develop genetic abnormalities.The study of XCI patterns suggest that, although in a limited number of cases, clonality can be observed in MSCs derived from MPN patients.Constitutional XCI skewing can be involved in the pathogenesis of malignancies, as it is thought to mediate susceptibility to breast and ovarian cancer.Our observation, however, deserves further confirmation, as the methylation of the androgen receptor does not always reflect chromosome X inactivation, as recently reported.In conclusion, our results lend support to the hypothesis that a primary MSC defect may either lead to or favor Ph-neg MPNs, and represent a particular mechanism of leukemogenesis in which both hematopoiesis and MSCs are altered in response to damage-inducing factors.How the presence and characteristics of the functional and genetic abnormalities reported here contribute to the different phenotypes in Ph-neg MPNs is a matter of further investigation.
