Characteristics of bone marrow fibroblast colony-forming cells (CFU-F) and their progeny in patients with myeloproliferative disorders

Abstract

Chronic myeloproliferative disorders (MPD) are clonal diseases of the pluripotent hematopoietic stem cell frequently associated with myelofibrosis (MF). There is only indirect evidence indicating that the increased deposition of collagen in bone marrow matrix is a secondary phenomenon. A liquid culture system for cloning and growing bone marrow fibroblasts has permitted us to approach more directly the understanding of the pathogenesis of myelofibrosis by comparing the biophysical, growth, and functional characteristics of fibroblasts from normals, MPD patients without MF, and those with MF. In patients with MF, marrow fibroblast colony (CFU-F) formation could not be studied; fibroblasts were grown from marrow explants. CFU-E from normals and MPD patients exhibited similar cell density distribution and similar cell sedimentation rates. These similarities contrasted sharply with the differences seen when the erythroid and granulocyte-macrophage progenitors were studied by the same methods. There was a marked light density shift and a rapidly sedimenting shift of MPD hematopoietic colony-forming cells. Marrow fibroblasts from MPD patients with and without MF displayed the same in vitro growth characteristics as fibroblasts from normals. Both types of fibroblasts exhibited anchorage and serum dependence, and contact inhibition of growth. Marrow fibroblasts were also characterized for the presence and distribution of fibronectin and collagen types by immunofluorescent staining using monospecific antibodies. Extracellular matrix, membrane-, and cytoplasm- associated fibronectin, type I, type III, and type V collagen showed a similar staining pattern in both normal and myelofibrotic marrow fibroblasts. Plasminogen-dependent fibrinolytic activity elicited from normal and myelofibrotic marrow fibroblasts were equivalent. Chromosomal analysis of hematopoietic cells and marrow fibroblasts from Philadelphia chromosome positive chronic myelocytic leukemia patients with and without MF showed that the Philadelphia chromosome was present only in hematopoietic cells. The results of these studies taken together demonstrate that bone marrow collagen-producing cells from MPD patients with and without MF behave in vitro as do those from normals. These findings support the hypothesis that that the marrow fibrosis observed in patients with MPD results from a reactive process rather than from a primary disorder affecting the marrow collagen-producing cells.

Circulating Osteogenic Precursor Cells in Heterotopic Bone Formation

“…The cell suspension was seeded into tissue culture flasks or plates at a density of 5 × 10 5 –1 × 10 6 cells/cm 2 , and in all comparisons, equal numbers of cells were used. After 3 days, nonadherent cells were removed, and the remaining adherent cells were refed twice weekly for colony‐forming assays [29] or establishment of primary cultures. Colonies containing >50 cells were selected using a cloning ring and single colony‐derived strains were passaged in α‐MEM + 15% FBS at confluency.…”

Inflammation as a Keystone of Bone Marrow Stroma Alterations in Primary Myelofibrosis

“…If evidences are still lacking to attest the nonclonal nature of stromal cells in PMF, some data must be discussed. Cytogenetic-based analyses of bone marrow fibroblasts or MSCs isolated from PMF patients are ancient and based on low-sensitive technics [ 119 – 121 ]. Recently, some data suggested that MSCs from patients could display cytogenetic modification, before culture [ 122 ].…”

The expression of lysyl‐oxidase gene family members in myeloproliferative neoplasms

“…This is particularly evident in primary myelofibrosis (PMF) but is also associated with disease progression in all the other MPNs entities . Fibrosis of the bone marrow is considered as a secondary phenomenon in MPNs, as fibroblasts are not part of the environment in these disorders . The fibrosis in these cases is probably linked to abnormal production and release of several cytokines and growth factors by hematopoietic cells, especially dystrophic megakaryocytes of the malignant clone These cytokines and growth factors include platelet‐derived growth factor (PDGF), transforming growth factor beta, basic fibroblast growth factor, vascular endothelial growth factor and platelet factor‐4 (PF‐4).…”