Ectopic Expression of CCAAT/Enhancer Binding Proteinβ(C/EBPβ) in Long-Term Bone Marrow Cultures Induces Granulopoiesis and Alters Stromal Cell Function

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.

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Sugar receptors of the stromal cell layer in human long-term bone marrow cultures: Their presence, modulatory responses to changes in the microenvironment and potential role in cellular adhesion

Blut Zeitschrift für die gesamte Blutforschung ◽

10.1007/bf01744137 ◽

1990 ◽

Vol 61 (4) ◽

pp. 232-239 ◽

Cited By ~ 12

Author(s):

Sigrun Gabius ◽

Hans-Joachim Gabius

Keyword(s):

Bone Marrow ◽

Stromal Cell ◽

Potential Role ◽

Cell Layer ◽

Cellular Adhesion ◽

Bone Marrow Cultures ◽

Sugar Receptors ◽

Marrow Cultures

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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 ◽

10.1182/blood.v68.6.1348.bloodjournal6861348 ◽

1986 ◽

Vol 68 (6) ◽

pp. 1348-1354 ◽

Cited By ~ 2

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.

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

Journal of Experimental Medicine ◽

10.1084/jem.171.2.477 ◽

1990 ◽

Vol 171 (2) ◽

pp. 477-488 ◽

Cited By ~ 369

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.

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Haemopoietic Inductive Capacity of Irradiated Stromal Cell Layers In Human Micro Long-Term Bone Marrow Cultures

Cell Proliferation ◽

10.1111/j.1365-2184.1988.tb00801.x ◽

1988 ◽

Vol 21 (6) ◽

pp. 411-417

Author(s):

P. Brühl ◽

H.-G. Mergenthaler ◽

P. Dörmer

Keyword(s):

Bone Marrow ◽

Stromal Cell ◽

Bone Marrow Cultures ◽

Cell Layers ◽

Marrow Cultures

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.4.7897180 ◽

1995 ◽

Vol 43 (4) ◽

pp. 371-379 ◽

Cited By ~ 13

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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