Dissecting the hematopoietic microenvironment. IX. Further characterization of murine bone marrow stromal cells

Abstract We have previously shown the adherent nontransformed, nonimmortalized murine bone marrow stromal cell (BMSC) population to consist of phagocytic macrophage and endothelial-like cells and nonphagocytic fibroblasts. Both colonial and near confluent growth of each cell type was obtained following magnetic bead separation, subsequent passaging, and sustained culture with fetal bovine serum and cytokines. Monoclonal antibody staining of antigenic determinants was used to characterize the phenotype of the stromal cell population in primary platings of murine colony-forming unit fibroblast and long-term bone marrow cultures. The antibodies MECA-99, MECA-32, and MJ7–18, raised against murine vascular endothelial antigenic determinants, and von Willebrand's factor all stained selectively for the rounded endothelial- like cells. Endothelial-like cells as well as macrophages expressed the myeloid surface antigens F4/80, 7/4, and Mac-1 under our culture conditions. The cytoskeleton of the stromal fibroblasts in culture was shown to express smooth muscle-specific actin isoforms, as evidenced by positive staining of stress fibers for alpha smooth muscle-1, CGA-7 (alpha/gamma isoforms), and HHF-35 (recognizes all muscle-specific actins). Under culture conditions, stromal fibroblasts were also found to be positive for a polyclonal smooth muscle myosin. It was found that these fibroblasts stained for collagens type I, III, and IV in our cultures. Although collagen type IV is considered a by-product of endothelial cells, endothelial-like cells in our cultures did not stain for any of the collagen types. We propose a classification listing for murine BMSCs as macrophages, endotheliallike cells, and fibroblasts that display smooth muscle-like characteristics in culture.

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Dissecting the hematopoietic microenvironment. IX. Further characterization of murine bone marrow stromal cells

Blood ◽

10.1182/blood.v81.5.1205.bloodjournal8151205 ◽

1993 ◽

Vol 81 (5) ◽

pp. 1205-1213

Author(s):

PE Penn ◽

DZ Jiang ◽

RG Fei ◽

E Sitnicka ◽

NS Wolf

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Stromal Cell ◽

Culture Conditions ◽

Antigenic Determinants ◽

Collagen Type Iv ◽

Positive Staining ◽

Type I ◽

Stromal Fibroblasts ◽

Murine Bone Marrow

We have previously shown the adherent nontransformed, nonimmortalized murine bone marrow stromal cell (BMSC) population to consist of phagocytic macrophage and endothelial-like cells and nonphagocytic fibroblasts. Both colonial and near confluent growth of each cell type was obtained following magnetic bead separation, subsequent passaging, and sustained culture with fetal bovine serum and cytokines. Monoclonal antibody staining of antigenic determinants was used to characterize the phenotype of the stromal cell population in primary platings of murine colony-forming unit fibroblast and long-term bone marrow cultures. The antibodies MECA-99, MECA-32, and MJ7–18, raised against murine vascular endothelial antigenic determinants, and von Willebrand's factor all stained selectively for the rounded endothelial- like cells. Endothelial-like cells as well as macrophages expressed the myeloid surface antigens F4/80, 7/4, and Mac-1 under our culture conditions. The cytoskeleton of the stromal fibroblasts in culture was shown to express smooth muscle-specific actin isoforms, as evidenced by positive staining of stress fibers for alpha smooth muscle-1, CGA-7 (alpha/gamma isoforms), and HHF-35 (recognizes all muscle-specific actins). Under culture conditions, stromal fibroblasts were also found to be positive for a polyclonal smooth muscle myosin. It was found that these fibroblasts stained for collagens type I, III, and IV in our cultures. Although collagen type IV is considered a by-product of endothelial cells, endothelial-like cells in our cultures did not stain for any of the collagen types. We propose a classification listing for murine BMSCs as macrophages, endotheliallike cells, and fibroblasts that display smooth muscle-like characteristics in culture.

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A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture.

The Journal of Cell Biology ◽

10.1083/jcb.114.3.557 ◽

1991 ◽

Vol 114 (3) ◽

pp. 557-565 ◽

Cited By ~ 246

Author(s):

K Miyake ◽

K Medina ◽

K Ishihara ◽

M Kimoto ◽

R Auerbach ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Cell Adhesion ◽

Endothelial Cell ◽

Adhesion Molecule ◽

Stromal Cells ◽

Stromal Cell ◽

Cell Adhesion Molecule ◽

B Lymphocyte ◽

Murine Bone Marrow

Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation.

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Proteoglycan synthesis in two murine bone marrow stromal cell lines

Blood ◽

10.1182/blood.v70.6.1777.bloodjournal7061777 ◽

1987 ◽

Vol 70 (6) ◽

pp. 1777-1783 ◽

Cited By ~ 1

Author(s):

SL Kirby ◽

SA Bentley

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Stromal Cell ◽

Dermatan Sulfate ◽

Cell Layer ◽

Gradient Centrifugation ◽

Exchange Chromatography ◽

Proteoglycan Synthesis ◽

Murine Bone Marrow

There is evidence indicating that stromal proteoglycans are an important functional component of the hematopoietic microenvironment. Proteoglycan synthesis was therefore investigated in the MS3–2A and D2XRII hematopoietic stromal cell lines. These lines differ in their capacity to support hematopoiesis in vitro, D2XRII supporting in vitro hematopoiesis, whereas MS3–2A does not. Cells were labeled with 35S- sulfate as precursor, and 4 mol/L guanidine HCl extracts of cells and media were analyzed by ion-exchange chromatography, cesium chloride density gradient centrifugation, and molecular sieve chromatography. Proteoglycans were further examined by enzymatic and chemical digestions. MS3–2A cells produced at least three proteoglycan species. Two chondroitin/dermatan sulfate (CS/DS) proteoglycans, Kav = 0.40 and Kav = 0.68 on Sepharose CL-2B, were present primarily in the medium. The respective glycosaminoglycan molecular weight (mol wt) values were 38 kd and 40 kd. A heparan sulfate (HS) proteoglycan of Kav = 0.58 and glycosaminoglycan mol wt 36 kd was present primarily in the cell layer extract. D2XRII cells synthesized two HS proteoglycans. The larger (Kav = 0.45; glycosaminoglycan mol wt, 30 kd) was of low density on gradient centrifugation and more prominent in the cell layer extracts, whereas the smaller (Kav = 0.68; glycosaminoglycan mol wt, 38 kd) was dense and present mainly in the culture medium. A single CS/DS proteoglycan species of Kav 0.78 and average glycosaminoglycan of mol wt 18 kd was present in roughly equal amounts in the medium and in the cell layer. MS3–2A and D2XRII thus appear phenotypically distinct with respect to proteoglycan synthesis. These differences are discussed in relation to the microenvironmental function of bone marrow stromal elements.

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Internalization of human immunodeficiency virus type I and other retroviruses by megakaryocytes and platelets

Blood ◽

10.1182/blood.v75.10.1920.1920 ◽

1990 ◽

Vol 75 (10) ◽

pp. 1920-1923 ◽

Cited By ~ 98

Author(s):

D Zucker-Franklin ◽

S Seremetis ◽

ZY Zheng

Keyword(s):

Bone Marrow ◽

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Target Cells ◽

Type I ◽

Murine Bone Marrow ◽

Immunodeficiency Virus ◽

Normal Human ◽

Normal Human Bone Marrow

Abstract Direct infection of megakaryocytes and platelets by human immunodeficiency virus type I (HIV-I) or other retroviruses has not been demonstrated. To determine whether this could occur, murine bone marrow was co-cultivated with the amphotropic retrovirus-producing cell line PA317-N2, and freshly isolated normal human bone marrow and platelets were co-cultivated with HIV-infected H9 cells. In each case, ultrastructural analyses showed viruses within megakaryocytes and platelets. In murine specimens, the uptake of retrovirus was avid at all stages of differentiation. In human specimens, viral uptake was less frequent. These results suggest that direct infection of megakaryocytes could play a role in the pathophysiology of HIV- associated disease. In addition, these observations suggest that cells of the megakaryocyte lineage could serve as target cells in gene transfer experiments using retroviral-based vectors.

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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.v68.6.1348.1348 ◽

1986 ◽

Vol 68 (6) ◽

pp. 1348-1354 ◽

Cited By ~ 53

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.

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