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.

scholarly journals Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Culture Conditions ◽  
B Lymphopoiesis ◽  
Bone Marrow Cultures ◽  
Factor Secretion ◽  
Marrow Cultures

Abstract Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.

scholarly journals Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Culture Conditions ◽  
B Lymphopoiesis ◽  
Bone Marrow Cultures ◽  
Factor Secretion ◽  
Marrow Cultures

Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.

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 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 Morphological characterization of stromal cell types in hematopoietically active long-term murine bone marrow cultures.

1995 ◽  
Vol 43 (4) ◽  
pp. 371-379 ◽  
Author(s):  
S P Hauser ◽  
J A Waldron ◽  
K B Upuda ◽  
D A Lipschitz
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Stromal Cell ◽  
Cell Types ◽  
Histological Evaluation ◽  
Type I ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Accurate histological evaluation of stromal morphology is very difficult in cultures incubated in plastic flasks. Employing glass flasketts, we were able to characterize the morphology and immunocytochemistry of four marrow stromal cell types in a functionally intact microenvironment of murine long-term bone marrow cultures (LTBMCs). Fibroblastoid cells stained positively for collagen Type I and III, negatively for von Willebrand factor (vWf), the mouse macrophage F4/80 antigen, and the Bandeiraea simplicifolia lectin I isolectin B4 (BSL I-B4). Endothelial cells stained positively for vWf antigen and lectin BSL I-B4 but negatively for collagen Types I and III and for F4/80 antigen. Fat-containing cells had a dense, ovaloid, indented nucleus and fat-containing vacuoles. Macrophages were strongly positive for the F4/80 antigen and stained weakly with BSL I-B4. Between the fourth and ninth weeks after culture initiation, fibroblastoid and endothelial cells remained constant, between 21 +/- 2% and 24 +/- 2% and between 3 +/- 0.3% and 4 +/- 0.4%, respectively, of the total stromal cell population. By contrast, the percentage of fat-containing cells decreased significantly from 26 +/- 3% at Week 4 to 17 +/- 2% at Week 9, and macrophages increased significantly from 49 +/- 1% at Week 4 to 57 +/- 1% at Week 9. This characterization of the stromal cell types in functionally intact LTBMCs should assist in the study of the complex interactions among the marrow stroma, cytokine production, and hematopoiesis.

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