Presence Of Bone Marrow Stromal Cells Research Articles

ONCOSTATIN M STIMULATES MACROPHAGE-POLYKARYON FORMATION IN LONG-TERM HUMAN BONE-MARROW CULTURES

Though oncostatin M (OSM) is a potent mediator of the inflammatory reaction, its role in inflammation and bone resorption is still unclear. A long-term bone-marrow culture system is usually developed to allow the formation of multinucleated cells (MNC) and was used here to define the effects of human recombinant OSM on human MNC formation. OSM significantly upregulated (1.9- to 5.6-fold) the number of MNC in these cultures in a dose- and time-dependent manner. Cell nucleation and tartrate-resistant acid phosphatase activity were also increased. MNC did not display osteoclast characteristics, such as response to calcitonin and failure to resorb dentin surface. However, they expressed a non-specific α-naphthyl acetate esterase as well as macrophage differentiation antigens (CD11b, CD13 and CD33) and were able to perform phagocytosis. Similar effects were observed after addition of 1α,25-dihydroxyvitamin D3. Moreover, in these culture conditions, human bone-marrow mononuclear cells were capable of low-grade resportion in the presence of bone-marrow stromal cells. This low grade resorption was significantly inhibited by addition of 25 ng/ml OSM. Our data demonstrate for the first time that human recombinant OSM significantly stimulates the formation of MNC and could be involved in the inflammatory process via macrophage-polykaryon formation from human bone marrow.

Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro

Leukaemic cells from most cases of B-chronic lymphocytic leukaemia die rapidly by apoptosis in vitro unless they are cultured in the presence of interleukin-4 or interferon alpha or gamma. We now report prolonged survival of purified CLL cells cultured on bone marrow (BM) derived stromal cells in the absence of exogenous growth factors. In 10 cases of CLL examined 0-61% (mean 14.7%) of the cells were viable after 10 d culture in medium alone, whereas in the presence of BM stromal cells 10-102% (mean 47.0%) of cells were recovered alive (P < 0.005) in 7/10 cases of CLL, cells remained viable after 30 d of culture in BM stromal cells with cell recovery of 12-65%. These long-term cultured CLL cells were Epstein Barr virus negative, shown by the failure to detect the ENBA-2 and BZLF1 genes of EBV by PCR analysis. Identity between day 0 and day 30 CLL cells was demonstrated by sequence analysis of their clonal IgH CDR3 region. Adherence of CLL cells to BM stromal cell layers was critical for their protection from apoptosis. Separation of CLL cells from stroma by 0.45 micron culture filters resulted in loss of the protective effect of the stromal cells. Stromal cells were also able to protect CLL cells from hydrocortisone-induced apoptotic cell death. Our findings provide an in vitro system that can be used to analyse the growth requirements of CLL cells and their chemosensitivity in an in vitro environment that mimics the in vivo milieu.

Bone marrow-derived stromal cells prevent apoptotic cell death in B- lineage acute lymphoblastic leukemia

Establishing requirements for the survival of human immature B cells in vitro has proved elusive. In this article, we report prolonged survival of B-lineage leukemic cells on ‘feeder layers’ of bone marrow (BM)- derived stromal cells in a serum-free environment. In 15 of 18 cases of B-lineage acute lymphoblastic leukemia (ALL), there was a greater than 50% decrease in the number of viable cells after 72 hours of culture in medium alone. Cell loss was preceded by molecular and cellular changes characteristic of programmed cell death, or apoptosis, and was not suppressed by adding interleukin-7 to the tissue culture medium. By contrast, the use of allogeneic BM stromal cells as feeder layers prevented apoptosis in 10 of 12 cases of ALL, leading to extended survival of the blast cells. This method was not successful when the allogeneic marrow cells were replaced with established cell lines. In six of eight cases in which the numbers of intact CD19+ lymphoblasts were counted by flow cytometry after 7 days of culture, the proportion of such cells recovered in the presence of BM stromal cells was 68.8% to 124.7% (median, 95.3%) of that originally seeded, as opposed to the 0.3% to 15.9% fraction (median, 0.7%) obtained in the absence of stromal cells. Survival requirements of the B-lineage lymphoblasts appeared to be heterogeneous, as cells from 3 of the 18 cases studied showed no signs of apoptosis in serum-free tissue culture medium that lacked BM stromal cells. The only cells not giving rise to viable cultures were from two hyperdiploid (greater than 50 chromosomes) cases with identical karyotypes. The serum-free assay described here can be used to compare the survival requirements of normal and leukemic B-cell progenitors as well as to identify the molecules involved in the interaction between BM stroma and immature B cells.

Bone Marrow-Derived Stromal Cells Prevent Apoptotic Cell Death in B-Lineage Acute Lymphoblastic Leukemia

Establishing requirements for the survival of human immature B cells in vitro has proved elusive. In this article, we report prolonged survival of B-lineage leukemic cells on 'feeder layers' of bone marrow (BM)-derived stromal cells in a serum-free environment. In 15 of 18 cases of B-lineage acute lymphoblastic leukemia (ALL), there was a greater than 50% decrease in the number of viable cells after 72 hours of culture in medium alone. Cell loss was preceded by molecular and cellular changes characteristic of programmed cell death, or apoptosis, and was not suppressed by adding interleukin-7 to the tissue culture medium. By contrast, the use of allogeneic BM stromal cells as feeder layers prevented apoptosis in 10 of 12 cases of ALL, leading to extended survival of the blast cells. This method was not successful when the allogeneic marrow cells were replaced with established cell lines. In six of eight cases in which the numbers of intact CD19+ lymphoblasts were counted by flow cytometry after 7 days of culture, the proportion of such cells recovered in the presence of BM stromal cells was 68.8% to 124.7% (median, 95.3%) of that originally seeded, as opposed to the 0.3% to 15.9% fraction (median, 0.7%) obtained in the absence of stromal cells. Survival requirements of the B-lineage lymphoblasts appeared to be heterogeneous, as cells from 3 of the 18 cases studied showed no signs of apoptosis in serum-free tissue culture medium that lacked BM stromal cells. The only cells not giving rise to viable cultures were from two hyperdiploid (greater than 50 chromosomes) cases with identical karyotypes. The serum-free assay described here can be used to compare the survival requirements of normal and leukemic B-cell progenitors as well as to identify the molecules involved in the interaction between BM stroma and immature B cells.