A Novel in Vitro Model of Focal Fibrosis in Bone Marrow Stromal Co-Culture of CD34+ Cells From Patients with Idiopathic Myelofibrosis.

Abstract 2896

Poster Board II-872

Chronic idiopathic myelofibrosis (IMF), essential thrombocythemia, polycythemia vera (PV) and chronic myelogenous leukemia (CML) are chronic myeloproliferative disorders (MPDs) characterized by the clonal proliferation of one or more hematopoietic lineage. In this study we investigated the interaction of MPDs stem/progenitor cells with bone marrow stroma. We established serum-free co-cultures of purified CD34+ cells from 59 peripheral blood (PB) samples from 30 MPD patients using the murine bone marrow stromal cell line OP9 transduced with an adenoviral vector expressing the human thrombopoietin gene (Adeno-Tpo). We observed that patients with IMF invariable had elevated numbers of CD34+ cells in PB, comparable to numbers reported in G-CSF mobilized PB (MPB). In co-cultures of CD34+cells from IMF PB or normal MPB there was an increase in total cells (IMF, 639±99 folds, N=31; MPB, 180±30, N=3) by 13-18 days with a high percentage expressing the megakaryocyte-platelet lineage marker CD41a (IMF 24-63%, MPB 18-35%) and <5% expressing CD14 (a monocytic marker). In co-culture of CD34+ cells from PV specimens, there was cellular expansion of 45±30 folds (N=3) but with <5% CD41a+ and >24 % CD14+ cells. CML PB cellular expansion was 147±45 folds (N=4) with 6-32 % CD41a+, 5-22% CD14+ and >60% CD15+ cells. In IMF stromal co-cultures, we observed the development of focal areas of OP9 stromal cell fibrosis (FF) at 12-14 days, associated with clonal proliferation of megakaryocyte progenitors and their differentiation to megakaryocytes and pro-platelets. This was associated with suppression of the spontaneous differentiation of OP9 cells to adipocytes. We obtained similar results in serum-free co-culture using the murine bone marrow stromal line MS-5 transduced with adeno-Tpo. This focal fibrosis phenomenon was not uniquely associated with IMF since we observed FF in normal MBP CD34+ co-culture in association with foci of magakaryocyte/platelet production, however the number of areas of FF was significantly higher when IMF CD34+ were compared to CD34+ cells from normal MPB or other types of MPDs. In IMF there were 84±17 FF/1,000 CD34+ (n=10), in PV <3 FF/1,000 (n=3) and in CML <9 FF/1,000 (n=3). Interferon α-2b is known to inhibit thrombopoietin-induced megakaryocytopoiesis and in clinical trials we have observed that IFNα-2b may retard progression of early IMF (Silver RT, Vandris K Leukemia 2009;23:1366). Addition of IFNα-2b to IMF CD34+ co-cultures dramatically reduced (>70%) total nucleated cells, CD41a+ cells, and FF-forming progenitor cells compared to control. This serum-free bone marrow stromal co-culture system distinguish different types of MPDs and may be useful in monitoring disease progression and patient response to therapy in IMF. The in vitro system appears to recapitulate the process of marrow fibrosis in the patient and can thus provide a sensitive bioassay to evaluate potential drugs for IMF therapy.