Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

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Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Blood ◽

10.1182/blood.v66.2.447.447 ◽

1985 ◽

Vol 66 (2) ◽

pp. 447-455 ◽

Cited By ~ 66

Author(s):

D Zipori ◽

J Toledo ◽

K von der Mark

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Cell Line ◽

Cell Lines ◽

Stromal Cell ◽

Leukemic Cells ◽

Matrix Composition ◽

Mouse Bone Marrow ◽

Type I

Abstract Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

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Stromal cell-associated hematopoiesis: immortalization and characterization of a primate bone marrow-derived stromal cell line

Blood ◽

10.1182/blood.v77.8.1723.1723 ◽

1991 ◽

Vol 77 (8) ◽

pp. 1723-1733 ◽

Cited By ~ 50

Author(s):

SR Paul ◽

YC Yang ◽

RE Donahue ◽

S Goldring ◽

DA Williams

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Cell Line ◽

Cell Lines ◽

Stromal Cell ◽

Bone Marrow Stromal Cell ◽

Hematopoietic Stem ◽

Stromal Cell Line

Abstract An elucidation of the interaction between the bone marrow microenvironment and hematopoietic stem cells is critical to the understanding of the molecular basis of stem cell self renewal and differentiation. This interaction is dependent, at least in part, on direct cell to cell contact or cellular adhesion to extracellular matrix proteins. Long-term bone marrow cultures (LTMC) provide an appropriate microenvironment for maintenance of primitive hematopoietic stem cells and a means of analyzing this stem cell-stromal cell interaction in vitro. Although LTMC have been successfully generated from murine and human bone marrow, only limited success has been reported in a primate system. In addition, few permanent stromal cell lines are available from nonmurine bone marrow. Because the primate has become a useful model for large animal bone marrow transplant studies and, more specifically, retroviral-mediated gene transfer analysis, we have generated immortalized bone marrow stromal cell lines from primate bone marrow using gene transfer of the Simian virus large T (SV40 LT) antigen. At least one stromal cell line has demonstrated the capacity to maintain early hematopoietic cells in long-term cultures for up to 4 weeks as measured by in vitro progenitor assays. Studies were undertaken to characterize the products of extracellular matrix biosynthesis and growth factor synthesis of this cell line, designated PU-34. In contrast to most murine bone marrow-derived stromal cell lines capable of supporting hematopoiesis in vitro that have been examined, the extracellular matrix produced by this primate cell line includes collagen types I, laminin. Growth factor production analyzed through RNA blot analysis, bone marrow cell culture data, and factor- dependent cell line proliferation assays includes interleukin-6 (IL-6), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, leukemia inhibitory factor, and a novel cytokine designated IL- 11. This immortalized primate bone marrow stromal cell line may be useful in maintaining early progenitor cells for experimental manipulation without the loss of reconstituting capacity and as a potential source of novel hematopoietic growth factors.

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Stromal cell-associated hematopoiesis: immortalization and characterization of a primate bone marrow-derived stromal cell line

Blood ◽

10.1182/blood.v77.8.1723.bloodjournal7781723 ◽

1991 ◽

Vol 77 (8) ◽

pp. 1723-1733 ◽

Cited By ~ 1

Author(s):

SR Paul ◽

YC Yang ◽

RE Donahue ◽

S Goldring ◽

DA Williams

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Cell Line ◽

Cell Lines ◽

Stromal Cell ◽

Bone Marrow Stromal Cell ◽

Hematopoietic Stem ◽

Stromal Cell Line

An elucidation of the interaction between the bone marrow microenvironment and hematopoietic stem cells is critical to the understanding of the molecular basis of stem cell self renewal and differentiation. This interaction is dependent, at least in part, on direct cell to cell contact or cellular adhesion to extracellular matrix proteins. Long-term bone marrow cultures (LTMC) provide an appropriate microenvironment for maintenance of primitive hematopoietic stem cells and a means of analyzing this stem cell-stromal cell interaction in vitro. Although LTMC have been successfully generated from murine and human bone marrow, only limited success has been reported in a primate system. In addition, few permanent stromal cell lines are available from nonmurine bone marrow. Because the primate has become a useful model for large animal bone marrow transplant studies and, more specifically, retroviral-mediated gene transfer analysis, we have generated immortalized bone marrow stromal cell lines from primate bone marrow using gene transfer of the Simian virus large T (SV40 LT) antigen. At least one stromal cell line has demonstrated the capacity to maintain early hematopoietic cells in long-term cultures for up to 4 weeks as measured by in vitro progenitor assays. Studies were undertaken to characterize the products of extracellular matrix biosynthesis and growth factor synthesis of this cell line, designated PU-34. In contrast to most murine bone marrow-derived stromal cell lines capable of supporting hematopoiesis in vitro that have been examined, the extracellular matrix produced by this primate cell line includes collagen types I, laminin. Growth factor production analyzed through RNA blot analysis, bone marrow cell culture data, and factor- dependent cell line proliferation assays includes interleukin-6 (IL-6), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, leukemia inhibitory factor, and a novel cytokine designated IL- 11. This immortalized primate bone marrow stromal cell line may be useful in maintaining early progenitor cells for experimental manipulation without the loss of reconstituting capacity and as a potential source of novel hematopoietic growth factors.

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Hemonectin mediates adhesion of engrafted murine progenitors to a clonal bone marrow stromal cell line from Sl/Sld mice

Blood ◽

10.1182/blood.v77.8.1691.1691 ◽

1991 ◽

Vol 77 (8) ◽

pp. 1691-1698 ◽

Cited By ~ 14

Author(s):

P Anklesaria ◽

JS Greenberger ◽

TJ Fitzgerald ◽

B Sullenbarger ◽

M Wicha ◽

...

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Cell Lines ◽

Stromal Cells ◽

Stromal Cell ◽

Cumulative Number ◽

Specific Staining ◽

Marrow Stromal Cell

Abstract Mutant Sl/Sld mice exhibit decreased marrow hematopoiesis. The defect is known to reside in the marrow microenvironment of these animals, which is reproduced in vitro by primary marrow explants as well as by cloned marrow stromal cell lines. Bone marrow progenitor cells are incapable of adhering to primary Sl/Sld stromal cells or cloned stromal cell lines derived from them to form cobblestone-islands and proliferate. The role of hemonectin, a marrow-specific adhesion protein in the defective hematopoiesis of the Sl/Sld mice, was studied. Indirect immunoperoxidase staining of marrow in situ from Sl/Sld mice showed little specific staining while specific staining was seen in a pericellular distribution in marrow from +/+ mice. Hemonectin expression in several cloned stromal cell lines from Sl/Sld mice was compared by immunoblotting with that in cloned stromal cell lines from normal +/+ littermates. Cell line Sld3, which has the least hematopoiesis supportive capacity in vitro, showed no detectable hemonectin by immunoblotting, while Sld1 and Sld2 showed detectable but greatly reduced amounts compared with normal +/+ 2.4, GBI/6, and D2XRII. Confluent cultures incubated with purified hemonectin and engrafted with enriched progenitors showed a significant increase in the cumulative number of cobbleston-islands and day 14 spleen colony- forming units (CFU-s) forming progenitors (39.15 +/- 3.6/dish; 16.3 +/- 3.1/dish, respectively), compared with untreated Sld3 cultures (cobblestone-islands 8.1 +/- 3.6/dish; CFU-s forming progenitors 8.8 +/- 0.05/dish). Hemonectin-mediated progenitor cell binding to the Sld3 stromal cells was specifically inhibited by antihemonectin but not by preimmune serum. These data support the role of hemonectin in early progenitor-stromal cell interactions.

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Hemonectin mediates adhesion of engrafted murine progenitors to a clonal bone marrow stromal cell line from Sl/Sld mice

Blood ◽

10.1182/blood.v77.8.1691.bloodjournal7781691 ◽

1991 ◽

Vol 77 (8) ◽

pp. 1691-1698

Author(s):

P Anklesaria ◽

JS Greenberger ◽

TJ Fitzgerald ◽

B Sullenbarger ◽

M Wicha ◽

...

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Cell Lines ◽

Stromal Cells ◽

Stromal Cell ◽

Cumulative Number ◽

Specific Staining ◽

Marrow Stromal Cell

Mutant Sl/Sld mice exhibit decreased marrow hematopoiesis. The defect is known to reside in the marrow microenvironment of these animals, which is reproduced in vitro by primary marrow explants as well as by cloned marrow stromal cell lines. Bone marrow progenitor cells are incapable of adhering to primary Sl/Sld stromal cells or cloned stromal cell lines derived from them to form cobblestone-islands and proliferate. The role of hemonectin, a marrow-specific adhesion protein in the defective hematopoiesis of the Sl/Sld mice, was studied. Indirect immunoperoxidase staining of marrow in situ from Sl/Sld mice showed little specific staining while specific staining was seen in a pericellular distribution in marrow from +/+ mice. Hemonectin expression in several cloned stromal cell lines from Sl/Sld mice was compared by immunoblotting with that in cloned stromal cell lines from normal +/+ littermates. Cell line Sld3, which has the least hematopoiesis supportive capacity in vitro, showed no detectable hemonectin by immunoblotting, while Sld1 and Sld2 showed detectable but greatly reduced amounts compared with normal +/+ 2.4, GBI/6, and D2XRII. Confluent cultures incubated with purified hemonectin and engrafted with enriched progenitors showed a significant increase in the cumulative number of cobbleston-islands and day 14 spleen colony- forming units (CFU-s) forming progenitors (39.15 +/- 3.6/dish; 16.3 +/- 3.1/dish, respectively), compared with untreated Sld3 cultures (cobblestone-islands 8.1 +/- 3.6/dish; CFU-s forming progenitors 8.8 +/- 0.05/dish). Hemonectin-mediated progenitor cell binding to the Sld3 stromal cells was specifically inhibited by antihemonectin but not by preimmune serum. These data support the role of hemonectin in early progenitor-stromal cell interactions.

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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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Interleukin-10 inhibits the osteogenic activity of mouse bone marrow

Blood ◽

10.1182/blood.v82.8.2361.2361 ◽

1993 ◽

Vol 82 (8) ◽

pp. 2361-2370 ◽

Cited By ~ 40

Author(s):

P Van Vlasselaer ◽

B Borremans ◽

R Van Den Heuvel ◽

U Van Gorp ◽

R de Waal Malefyt

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Osteogenic Differentiation ◽

Interleukin 10 ◽

Mouse Bone Marrow ◽

Type I ◽

Mrna Levels ◽

Differentiation Potential ◽

Alp Activity ◽

Bone Proteins

Abstract Murine bone marrow cells synthesize bone proteins, including alkaline phosphatase (ALP), collagen type I, and osteocalcin, and form a mineralized extracellular matrix when cultured in the presence of beta- glycerophosphate and vitamin C. Interleukin-10 (IL-10) suppressed the synthesis of these bone proteins and mineralization without affecting cell proliferation. In addition, mRNA levels for the latter proteins were reduced in IL-10-treated cultures. This inhibitory effect was most outspoken when IL-10 was added before ALP activity peaked, eg, day 15 of culture. No significant effect was observed when IL-10 was added at later time points. This finding suggests that IL-10 acts at osteogenic differentiation stages that precede ALP expression but is ineffective on cells that progressed beyond this maturation stage. Likewise, IL-10 appeared to be unable to block both ALP activity and collagen synthesis in the preosteosteoblastic cell lines MN7 and MC3T3 that constitutively synthesize these proteins. Whereas IL-10 did not alter the number of fibroblast colony-forming cells of the marrow, it significantly reduced their osteogenic differentiation potential. In contrast to control cultures, IL-10-treated stroma was unable to either synthesize osteocalcin or to mineralize when subcultured over a 25-day period in the absence of IL-10. The inhibitory activity of IL-10 coincided with significant changes in stroma morphology. Whereas control cultures contained mainly flat adherent polygonal cells, significant numbers of rounded semiadherent to nonadherent cells were observed in the presence of IL-10. Scanning and transmission electron microscopy showed that, in contrast to control cultures, IL-10-treated stromas completely lacked a mineralized extracellular matrix. Collectively, these data suggest that IL-10 may have important regulatory effects on bone biology because of its capacity to downregulate early steps of osteogenic differentiation.

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In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line

Stem Cells International ◽

10.1155/2018/9896142 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Hong Kiat Lim ◽

Pravin Periasamy ◽

Helen C. O’Neill

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Stromal Cell ◽

Hematopoietic Cells ◽

Model Systems ◽

Hematopoietic Stem ◽

Cell Production ◽

Stromal Cell Line ◽

Reticular Cells

There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.

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