scholarly journals Role of colony-stimulating activity in murine long-term bone marrow cultures: evidence for its production and consumption by the adherent cells

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 761-767 ◽  
Author(s):  
JM Heard ◽  
S Fichelson ◽  
B Varet
Keyword(s):  
Bone Marrow ◽  
Myeloid Cells ◽  
Target Cells ◽  
Adherent Cells ◽  
Colony Stimulating Activity ◽  
Large Numbers ◽  
Bone Marrow Cultures ◽  
Nonadherent Cells ◽  
Marrow Cultures

Abstract The involvement of colony-stimulating activity (CSA) in murine long- term bone marrow cultures (LTBMC) was studied using bilayer agar cultures. The supernatants of LTBMC were removed, a layer of dense agar was spread over the cells adherent to the bottom of the flask, and fresh myeloid cells were plated as source of CFU-C in an upper agar layer. Large numbers of granulocytic and macrophagic colonies developed regularly when target cells were plated over adherent cells of nonrecharged and greater than 12 wk old LTBMC that were hematopoietically inactive (i.e., producing a low number of nonadherent cells). The removal of adherent cells from the myeloid cells used as source of CFU-C did not decrease the number of colonies. This suggests that adherent cells of LTBMC release CSA that is directly active on CFU- C. This CSA was no longer detectable over adherent layers of hematopoietically active LTBMC. A close inverse relationship was demonstrated between the number of nonadherent cells harvested before the assay and the level of CSA. No inhibitor for CSA was demonstrated in the supernatant of hematopoietically active cultures. Murine exogenous CSA incubated over the adherent layer host its activity within 24 hr, whereas in the same conditions human CSA retained its activity. These data demonstrate the production of CSA by the adherent layer of LTBMC and strongly suggest its specific in situ consumption by differentiating myeloid cells.

scholarly journals Role of colony-stimulating activity in murine long-term bone marrow cultures: evidence for its production and consumption by the adherent cells

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 761-767
Author(s):  
JM Heard ◽  
S Fichelson ◽  
B Varet
Keyword(s):  
Bone Marrow ◽  
Myeloid Cells ◽  
Target Cells ◽  
Adherent Cells ◽  
Colony Stimulating Activity ◽  
Large Numbers ◽  
Bone Marrow Cultures ◽  
Nonadherent Cells ◽  
Marrow Cultures

The involvement of colony-stimulating activity (CSA) in murine long- term bone marrow cultures (LTBMC) was studied using bilayer agar cultures. The supernatants of LTBMC were removed, a layer of dense agar was spread over the cells adherent to the bottom of the flask, and fresh myeloid cells were plated as source of CFU-C in an upper agar layer. Large numbers of granulocytic and macrophagic colonies developed regularly when target cells were plated over adherent cells of nonrecharged and greater than 12 wk old LTBMC that were hematopoietically inactive (i.e., producing a low number of nonadherent cells). The removal of adherent cells from the myeloid cells used as source of CFU-C did not decrease the number of colonies. This suggests that adherent cells of LTBMC release CSA that is directly active on CFU- C. This CSA was no longer detectable over adherent layers of hematopoietically active LTBMC. A close inverse relationship was demonstrated between the number of nonadherent cells harvested before the assay and the level of CSA. No inhibitor for CSA was demonstrated in the supernatant of hematopoietically active cultures. Murine exogenous CSA incubated over the adherent layer host its activity within 24 hr, whereas in the same conditions human CSA retained its activity. These data demonstrate the production of CSA by the adherent layer of LTBMC and strongly suggest its specific in situ consumption by differentiating myeloid cells.

scholarly journals Proteoglycans in human long-term bone marrow cultures: biochemical and ultrastructural analyses

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1333-1343 ◽  
Author(s):  
TN Wight ◽  
MG Kinsella ◽  
A Keating ◽  
JW Singer
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Layer ◽  
Ruthenium Red ◽  
Adherent Cells ◽  
Sulfate Proteoglycan ◽  
Chondroitinase Abc ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing 35S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCI and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (MI, CI) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) approximately 38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (MII, CII) that was included on Sepharose CL-4B at Kav = 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (MIIa, CIIa) and an included peak eluting at Kav = 0.39 (MIIb, CIIb). The void peaks (MIIa, CIIa) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroitinase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt approximately 38,000. The included peaks on Sepharose CL-6B (MIIb, CIIb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt approximately 38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chondrotinase AC (approximately 60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only approximately 10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H- activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of approximately 70% of the 35S-sulfate-labeled material from the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2269-2277 ◽  
Author(s):  
HM Lokhorst ◽  
T Lamme ◽  
M de Smet ◽  
S Klein ◽  
RA de Weger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Adherent Cells ◽  
Myeloma Cells ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA- 5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.

scholarly journals Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2269-2277 ◽  
Author(s):  
HM Lokhorst ◽  
T Lamme ◽  
M de Smet ◽  
S Klein ◽  
RA de Weger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Adherent Cells ◽  
Myeloma Cells ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA- 5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.

scholarly journals Proteoglycans in human long-term bone marrow cultures: biochemical and ultrastructural analyses

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1333-1343 ◽  
Author(s):  
TN Wight ◽  
MG Kinsella ◽  
A Keating ◽  
JW Singer
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Layer ◽  
Ruthenium Red ◽  
Adherent Cells ◽  
Sulfate Proteoglycan ◽  
Chondroitinase Abc ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing 35S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCI and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (MI, CI) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) approximately 38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (MII, CII) that was included on Sepharose CL-4B at Kav = 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (MIIa, CIIa) and an included peak eluting at Kav = 0.39 (MIIb, CIIb). The void peaks (MIIa, CIIa) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroitinase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt approximately 38,000. The included peaks on Sepharose CL-6B (MIIb, CIIb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt approximately 38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chondrotinase AC (approximately 60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only approximately 10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H- activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of approximately 70% of the 35S-sulfate-labeled material from the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1211-1217 ◽  
Author(s):  
DA Lipschitz ◽  
KB Udupa ◽  
JM Taylor ◽  
RK Shadduck ◽  
A Waheed
Keyword(s):  
Bone Marrow ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Colony Stimulating Factor ◽  
Other Substances ◽  
Bone Marrow Cultures ◽  
Medium Change ◽  
Stimulating Factor ◽  
Marrow Cultures

Abstract Weekly medium change or midweek feeding of long-term bone marrow cultures (LTMBCs) results in a significant increase in total myeloid cell production. Proliferative myeloid cells peak 48 hours after feeding, and nonproliferative myeloid cells reach maximum levels at 72 hours. This increase in myelopoiesis is invariably preceded by a significant elevation in biologically and immunologically measurable colony-stimulating factor (CSF) in the supernatants of LTBMC. The level peaks 24 hours after medium change, then gradually returns to basal values. The decrease in CSF relates to its consumption by generating myeloid precursors because no fluctuation in the levels occur in cultures without active myelopoiesis. No significant inhibitors or promoters of CSF were detected. When highly purified L cell CSF, CSF in lung-conditioned medium, or CSF concentrated from LTBMC supernatant is added to cultures, an identical increase in myelopoiesis occurs. Anti- CSF antiserum, added to culture at the time of medium change, totally neutralizes supernatant CSF levels but does not affect myelopoiesis. These findings suggest a potential regulatory role for CSF in myelopoiesis in LTBMC. CSF appears to function within the microenvironment through a mechanism involving cell:cell interactions or by causing the production of other substances that stimulate myelopoiesis. Because exogenous CSF stimulates myelopoiesis, it is likely that it too can react either directly or through microenvironmental cells to stimulate primitive myeloid cells to divide.

scholarly journals Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures.

1990 ◽  
Vol 171 (2) ◽  
pp. 477-488 ◽  
Author(s):  
K Miyake ◽  
K L Medina ◽  
S Hayashi ◽  
S Ono ◽  
T Hamaoka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cell ◽  
Myeloid Cells ◽  
Lymphoid Cells ◽  
Adhesion Assay ◽  
Bone Marrow Cultures ◽  
B Lineage ◽  
Biochemical Analyses ◽  
Marrow Cultures

A new panel of mAbs was prepared to a stromal cell line known to support lymphocytes in Whitlock-Witte type long-term bone marrow cultures. These antibodies were then screened with a cell adhesion assay and four were selected that inhibited the binding of B lineage cells to stromal cell monolayers. Immunofluorescent and biochemical analyses revealed that these new antibodies detected epitopes of the previously described Pgp-1/CD44 antigen complex. Addition of Pgp-1/CD44 antibodies to Dexter-type long-term bone marrow cultures completely prevented emergence of myeloid cells and they also blocked lymphocyte growth in Whitlock-Witte type cultures. mAbs MEL-14, LFA-1, and CD45R did not inhibit under the same conditions and there was no apparent relationship to Ig isotype. Adherent layers in treated cultures were not unusual in terms of morphology and the antibodies did not affect factor-dependent replication of lymphoid or myeloid progenitor cells. Therefore, the mechanism of inhibition may not involve direct toxicity to precursors or microenvironmental elements. Previous studies in humans and mice have implicated Pgp-1/CD44-related glycoproteins in the migration of peripheral lymphoid cells, as well as interactions of cells with the extracellular matrix. These findings suggest that they may also be critical for formation of lymphoid and myeloid cells within bone marrow.

Effects of bacterial lipopolysaccharide on the production of colony-stimulating activity in C3H/HeJ mouse long-term bone marrow cultures

1985 ◽  
Vol 9 (10) ◽  
pp. 1277-1282 ◽  
Author(s):  
Shin Aizawa ◽  
Masahito Tsurusawa ◽  
Takeshi Miyanomae ◽  
Hiroko Izumi ◽  
Kazuhiro J. Mori
Keyword(s):  
Bone Marrow ◽  
Bacterial Lipopolysaccharide ◽  
Colony Stimulating Activity ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Increased Adipocytogenesis and Hematopoiesis in Long-Term Bone Marrow Cultures from SMAD3−/− Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1298-1298
Author(s):  
Shaonan Cao ◽  
Shuanhu Zhou ◽  
Michael W. Epperly ◽  
Julie Glowacki ◽  
Donna Shields ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Bone Marrow ◽  
Adipocyte Differentiation ◽  
Irradiation Damage ◽  
Late Response ◽  
Adherent Cells ◽  
Receptor Signal ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract The abrogation of an intact TGFß/TGFß receptor signal transduction pathway in SMAD3−/− mice is associated with a decreased inflammatory response, and decreased acute and late response to ionizing irradiation damage. We tested the role of SMAD3 in bone marrow cellular capacity for hematopoiesis and in marrow stromal cellular potential for adipocytogenesis. Long-term bone marrow cultures (LTBMCs) were established from SMAD3−/−, +/−, and +/+ mice. Permanent clonal bone marrow stromal cell (MSC) lines were established from each mouse strain. The SMAD3+/+ and SMAD3+/− LTBMCs generated hematopoietic progenitor cells (by CFU-GEMM assay), and maintained active cobblestone island numbers and production of non-adherent cells for 22–23 weeks. In striking contrast, the SMAD3−/− LTBMCs demonstrated a significant increase in longevity of hematopoiesis as measured by each assay for over 34 weeks and non-adherent cells continued to form GEMM beyond 34 weeks. The number of CFU-GEMM per 5x104 cells (d 14) in SMAD3−/− cultures (188 ± 8.7) was 1250% greater (p<0.0001) than in SMAD3+/+ cultures (15.3 ± 2.3). Microscopic inspection at 27 weeks showed that more (16.5%, p<0.001) SMAD3−/− adherent cells contained lipid droplets (6.6% ± 0.7) compared with SMAD3+/+ adherent cells (0.4% ± 0.2). Because of that difference, we examined the regulation of adipocyte differentiation in MSC lines from those mice. First, there was more extensive (37-fold) adipocyte differentiation in SMAD3−/ − than SMAD3+/+ cultures. Second, there was a 7-fold attenuation of TGF-ß inhibition of adipocytogenesis in cells lacking SMAD3. RT-PCR evaluation showed greater expression of the adipocyte gene, ADIPSIN, and absence of its downregulation by TGFß in SMAD3−/− compared to SMAD3+/+. The data suggest that SMAD3 is a critical inhibitor of adipocyte differentiation. The data document pleiotrophic effects of interruption of the TGFß/TGFß receptor signal transduction cascade for both inhibition of hematopoietic support capacity and adipocyte differentiation of the bone marrow microenvironment.

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