Isolation, Expansion and Characterization of BM-Mesenchimal Cells (MSCs) in Patients Affected By Chronic Myeloproliferative neoplasm (MPN)

Introduction MSCs constitute a pivotal cell type capable of shaping both the architecture of the microenvironment and modulating communication between the various cell types through effects on the extracellular matrix (ECM) and by secretion of various growth factors and cytokines. MSCs and hematopoietic stem cells are thought to share the same mesenchymal origin. Some data confirm that MSCs express a functional erythropoietin receptor and JAK2-transduction pathway, but their role in the development and evolution of MPN is still not well known so our aim was the isolation, expansion and characterization of MSCs in patients affected by MPN. Some data indicates that BM-MSCs of patients affected by MPN do not carry the JAK2-V617F mutation. We studied 20 patients affected by MPN with the following characteristics: M/F 12/8, median age 53years, 8 affected by PV, 8 by ET and 4 by PMF. 15 patients were positive for JAK2 V617F mutation, 1 pts for cMPL and 2 were CARL mutations carriers.

Methods: MSC were isolated by bone marrow fraction by gradient separation on Lympholyte cell separation media and expanded in culture with a specific medium (MesenCult) in plastic-adherent cultures up to the second passage. DNA was extracted from MSC using QIAmp DNA Mini kit and the study of recurrent alterations (JAK2, MPL and CARL gene mutations was performed).

Results: MCS were expanded in 14 on 21 patients. Flow citometry analysis confirmed the standard MSC phenotype (CD45 negative, CD73 positive, CD90 positive and CD105 positive). The molecular analysis of JAKV617F, cMPL and CARL mutations resulted negative in all analyzed samples both in patients carriers of mutations and in wild type ones.

Conclusions: We conclude that common mutations markers of MPN neoplasm are absent in the mesenchymal compartment of bone marrows of patients affected by MPN and are restricted to the neoplastic clone.

This research project was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.)