Stromal cell-associated hematopoiesis: immortalization and characterization of a primate bone marrow-derived 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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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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Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

Blood ◽

10.1182/blood.v104.11.2328.2328 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2328-2328

Author(s):

Katja C. Weisel ◽

Ying Gao ◽

Jae-Hung Shieh ◽

Lothar Kanz ◽

Malcolm A.S. Moore

Keyword(s):

Bone Marrow ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Stromal Cells ◽

Stromal Cell ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Stromal Cell Line ◽

Mes Cells

Abstract The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

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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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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.bloodjournal662447 ◽

1985 ◽

Vol 66 (2) ◽

pp. 447-455

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

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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Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit

Blood ◽

10.1182/blood.v81.2.365.bloodjournal812365 ◽

1993 ◽

Vol 81 (2) ◽

pp. 365-372 ◽

Cited By ~ 63

Author(s):

JP Wineman ◽

S Nishikawa ◽

CE Muller-Sieburg

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Line ◽

Stromal Cells ◽

Stromal Cell ◽

Hematopoietic Stem ◽

Stem Cell Maintenance ◽

Stromal Cell Line ◽

Cell Maintenance

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

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Improved hematopoiesis in anemic Sl/Sld mice by splenectomy and therapeutic transplantation of a hematopoietic microenvironment

Blood ◽

10.1182/blood.v74.3.1144.bloodjournal7431144 ◽

1989 ◽

Vol 74 (3) ◽

pp. 1144-1151

Author(s):

P Anklesaria ◽

TJ FitzGerald ◽

K Kase ◽

A Ohara ◽

JS Greenberger

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Hind Limb ◽

Stromal Cell ◽

Bone Marrow Stromal Cell ◽

Volume Distribution ◽

Distribution Analysis ◽

Marrow Stromal Cell ◽

Stromal Cell Line ◽

The Right

The ability of a clonal hematopoiesis-supportive bone-marrow stromal cell line GBlneor to engraft and alter the microenvironment-induced anemia of Sl/Sld mice was studied. Prior to stromal cell transplantation, Sl/Sld mice received 1 Gy total body irradiation (TBI) and 13 Gy to the right hind limb. Two months after intravenous (IV) injection of 5 x 10(5) GBlneor cells, 54.4% +/- 17.0% donor origin (G418r) colony-forming cells were recovered from the right hind limb of Sl/Sld mice. Long-term bone marrow cultures (LTBMCs) established from GBlneor-transplanted mice produced 189.5 CFU-GEMM-forming progenitors/flask over 10 weeks compared with 52.7 +/- 6.2 CFU-GEMM forming progenitors/flask from irradiated nontransplanted Sl/Sld mice. A partial correction of macrocytic anemia was detected 2 months after GBlneor transplantation in splenectomized, irradiated Sl/Sld mice (HgB 7.2 +/- 0.4 g/dL; MCV 68.3 +/- 7.0 fL) compared to splenectomized, irradiated, nontransplanted Sl/Sld mice (HgB 5.5 +/- 1.1 g/dL; MCV 76 +/- 8.5 fL) or control Sl/Sld mice (HgB 5.4 +/- 0.5 g/dL; MCV 82.4 +/- 1.3 fL). Mean RBC volume distribution analysis showed a 2.5-fold increase in percentage of peripheral blood RBCs with MCV less than or equal to 45 fL and confirmed reduction of the MCV in splenectomized- GBlneor-transplanted mice compared to control Sl/Sld mice. A hematopoiesis-suppressive clonal stromal cell line derived from LTBMCs of Sl/Sld mice (Sldneor) engrafted as effectively (43.5% +/- 1.2% G418r CFU-F/limb) as did GBlneor cells (38.3% +/- 0.16% G418r CFU-F/limb) to the irradiated right hind limbs of C57Bl/6 mice. LTBMCs established after 2 or 6 months from Sldneor-transplanted mice showed decreased hematopoiesis (182 +/- 12 [2 months] and 3494.3 +/- 408.1 [6 months] CFU-GEMM forming progenitors/flask over 10 weeks) compared to those established from GBlneor-transplanted mice (5980 +/- 530 [2 months] and 7728 +/- 607, [6 months] CFU-GEMM progenitors forming/flask). Thus, transplantation of clonal bone-marrow stromal cell lines in vivo can stably transfer their physiologic properties to normal or mutant mice.

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