H Castro-Malaspina

The bone marrow stromal cell population is comprised of fibroblasts, endothelial cells, fat cells and “reticular cells.” The characteristics and the regulatory role in hematopoiesis of each of these cell types are unknown. A liquid culture system has been used to clone and to characterize human bone marrow fibroblast colony-forming cells (CFU-F). The linear relationship between the number of cells plated and the number of colonies formed suggests that fibroblast colonies originate in a single cell. Bone marrow CFU-F were adherent and nonphagocytic. The majority (90% ± 2%) were less dense than 1.070 g/cu cm. Velocity sedimentation separation demonstrated a heterogeneous CFU-F sedimentation rate, with a modal sedimentation of 4.95 ± 0.15 mm/hr. Analysis of CFU-F proliferative status by the thymidine suicide technique indicated that this cell was noncycling in individuals with undisturbed bone marrow function. Some of the more distinctive products of fibroblasts, other stromal cells, and hematopoietic colony-forming cells were used as positive and negative markers for CFU-F and the cells derived from them in vitro. Complement-mediated cytotoxicity using anti-Ia and anti-factor-VIII antigen antisera did not inhibit fibroblast colony formation. In contrast, a striking reduction of granulocyte-macrophage colony formation (CFU-c) was seen when bone marrow cells were treated with anti-Ia antiserum. Immunofluorescence staining was used to characterize the cells derived from CFU-F in vitro. No staining was observed after incubation of subconfluent cultures with anti-Ia and anti-factor-VIII antigen antisera. A positive immunofluorescent staining was obtained when isolated antibodies against three of the main proteins of bone marrow matrix: type I collagen, type III collagen, and fibronectin were used. Ultrastructural analysis showed that CFU-F progeny, in contrast to endothelial cells, did not contain Weibel-Palade bodies. These data support the conclusion that the colonies described in this study are of fibroblastic nature.

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.

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.