scholarly journals Proteoglycan synthesis in haematopoietic cells: isolation and characterization of heparan sulphate proteoglycans expressed by the bone-marrow stromal cell line MS-5

1997 ◽  
Vol 327 (2) ◽  
pp. 473-480 ◽  
Author(s):  
Zofia DRZENIEK ◽  
Barbara SIEBERTZ ◽  
Georg STÖCKER ◽  
Ursula JUST ◽  
Wolfram OSTERTAG ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Heparan Sulphate ◽  
Isolation And Characterization ◽  
Stromal Cell Line ◽  
Heparan Sulphate Proteoglycans

Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the haematopoietic microenvironment. Recently, several studies have indicated that they are involved in the interaction of haematopoietic stem and stromal cells. However, a detailed characterization of the heparan sulphate proteoglycans synthesized by bone-marrow stromal cells is still lacking. Here we report on the isolation and characterization of proteoglycans from the haematopoietic stromal cell line MS-5, that efficiently supports the growth and differentiation of human and murine haematopoietic progenitor cells. Biochemical characterization of purified proteoglycans revealed that the haematopoietic stromal cell line MS-5 synthesizes, in addition to chondroitin sulphate proteoglycans, several different heparan sulphate proteoglycans. Immunochemical analysis, using specific antibodies against the different members of the syndecan family, glypican, betaglycan and perlecan, showed that MS-5 cells synthesize all these different heparan sulphate proteoglycans. These data were further supported by reverse-transcriptase PCR and confirmed by sequence and Northern blot analysis. The relative abundance of the different heparan sulphate proteoglycans was estimated on the protein and mRNA levels.

scholarly journals Connexin-43-type gap junctions mediate communication between bone marrow stromal cells

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 38-45 ◽  
Author(s):  
K Dorshkind ◽  
L Green ◽  
A Godwin ◽  
WH Fletcher
Keyword(s):  
Bone Marrow ◽  
Gap Junctions ◽  
Cell Line ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Cell Communication ◽  
Marrow Stromal Cells ◽  
Stromal Cell Line ◽  
Cell Cell

Several morphologic studies have suggested that gap junctions exist between bone marrow stromal cells. This possibility was examined by analysis of stromal cells present in the adherent layer of primary long- term lymphoid bone marrow cultures and in additional studies using a stromal cell line. Results showing that the fluorescent dye lucifer yellow, when microinjected into a single stromal cell, transferred between most other contacting stroma and that stromal cells were electronically coupled provided support that cell-cell communication occurs between these microenvironmental elements. Additional studies showed that transcripts for connexin (Cx) 43, but not for Cx26 or Cx32, were present in a stromal cell line. To examine the potential for regulated cell-cell communication between the stroma, cells were treated with interleukin-1 (IL-1), a cytokine known to affect stromal cell function, and the effects on dye transfer were examined. IL-1 treatment resulted in a reversible decrease in the ability of dye to transfer between stromal cells in contact. Taken together, these studies show that gap junctions exist between stromal cells and that their permeability can be regulated. However, gap junction-mediated cell-cell communication could not be shown between the stroma and developing lymphoid cells.

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

Blood ◽  
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.

scholarly journals Proteoglycan synthesis in human and murine haematopoietic progenitor cell lines: isolation and characterization of a heparan sulphate proteoglycan as a major proteoglycan from the human haematopoietic cell line TF-1

1996 ◽  
Vol 317 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Georg STÖCKER ◽  
Zofia DRZENIEK ◽  
Ursula JUST ◽  
Wolfram OSTERTAG ◽  
Barbara SIEBERTZ ◽  
...  
Keyword(s):  
Cell Line ◽  
Stromal Cells ◽  
Progenitor Cell ◽  
Heparan Sulphate ◽  
Proteoglycan Synthesis ◽  
Heparan Sulphate Proteoglycan ◽  
Haematopoietic Progenitor Cell ◽  
Sulphate Proteoglycan ◽  
Isolation And Characterization

Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the microenvironment of haematopoietic tissues. Proteoglycans might also be involved in the interaction of haematopoietic stem and stromal cells. Recently, several studies have been reported on the proteoglycan synthesis of stromal cells, but little is known about the proteoglycan synthesis of haematopoietic stem or progenitor cells. Here we report on the isolation and characterization of proteoglycans from two haematopoietic progenitor cell lines, the murine FDCP-Mix A4 and the human TF-1 cell line. Proteoglycans were isolated from metabolically labelled cells and purified by several chromatographic steps, including anion-exchange and size-exclusion chromatography. Biochemical characterization was performed by electrophoresis or gel-filtration chromatography before and after digestion with glycosaminoglycan-specific enzymes or HNO2 treatment. Whereas FDCP-Mix A4 cells synthesize a homogeneous chondroitin 4-sulphate proteoglycan, isolation and characterization of proteoglycans from the human cell line TF-1 revealed, that TF-1 cells synthesize, in addition to a chondroitin sulphate proteoglycan, a heparan sulphate proteoglycan as major proteoglycan. For this heparan sulphate proteoglycan a core protein size of approx. 59 kDa was determined. Immunochemical analysis of this heparan sulphate proteoglycan revealed that it is not related to the syndecan family nor to glypican.

scholarly journals Differentiation of early plasma cells on bone marrow stromal cells requires interleukin-6 for escaping from apoptosis

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 487-494 ◽  
Author(s):  
MM Kawano ◽  
K Mihara ◽  
N Huang ◽  
T Tsujimoto ◽  
A Kuramoto
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Interleukin 6 ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Direct Evidence ◽  
Plasma Cells ◽  
Stromal Cell Line ◽  
Culture Supernatants

The bone marrow (BM) is well known to be the major site of Ig production in secondary immune responses; thus, the microenvironment of BM is considered to be essential for final differentiation of plasma cells. We identified in the peripheral blood (PB) early plasma cells (CD38++CD19+VLA-5-) committed to entering the BM. The sorted early plasma cells rapidly entered apoptosis in vitro, but these cells could survive and further differentiate into mature plasma cells (CD38 CD19+) just as BM plasma cells in the presence of a BM-derived stromal cell line (KM-102). Culture supernatants of KM-102 cell lines could also support survival of these cells, and antibody to interleukin-6 (IL-6) completely blocked the effect of these supernatants. Furthermore, recombinant IL-6, but not IL-1 or IL-3, could support their survival and their differentiation into mature plasma cells (CD38 CD19+VLA-5+) with expression of VLA-5 mRNA. Therefore, here is direct evidence that early plasma cells found in the PB differentiated into mature plasma cells with stromal cell-derived IL-6 in vitro; thus, BM stromal cells control the final checkpoint of plasma cell differentiation with secretion of IL-6 in the BM.

scholarly journals Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38- long-term culture-initiating cells

Blood ◽  
1994 ◽  
Vol 84 (12) ◽  
pp. 4116-4124 ◽  
Author(s):  
L Croisille ◽  
I Auffray ◽  
A Katz ◽  
B Izac ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Adherent Cells ◽  
Long Term Culture ◽  
Bone Marrow Cultures ◽  
Stromal Cell Line ◽  
Clonogenic Cells

Very primitive human hematopoietic progenitor cells are identified indirectly by their ability to give rise to clonogenic progenitors in the presence of either human or murine stromal cells. These long-term culture-initiating cell (LTC-IC) assays are usually performed in the presence of hydrocortisone based on the initial observation that hydrocortisone was required for prolonged hematopoiesis in standard long-term bone marrow cultures. In this report, we investigated the role of hydrocortisone in LTC-IC assays initiated with CD34++/CD38- cells seeded onto either human bone marrow LTC-derived adherent cells or a murine marrow-derived stromal cell line, MS-5. It was found that weekly addition of hydrocortisone to the cultures reduced the frequency of LTC-IC (from 1/5 to 1/20) calculated from limiting dilution experiments and also reduced fivefold to 10-fold the number of their progeny clonogenic cells detected after 4 to 5 weeks. In contrast, the frequency and differentiative potential of CD34++/CD38- grown in the presence of human marrow feeders was unaltered by the addition of glucocorticoids. Data are consistent with the hypothesis that hydrocortisone inhibited LTC-IC differentiation by downregulating the expression of a synergistic factor produced by MS-5 cells. (1) In the absence of hydrocortisone, the number of clonogenic progenitors generated by LTC-IC was much higher in cultures seeded on MS-5 than in cultures seeded on human marrow adherent cells, which was also true when cytokines were added to the cocultures. However, based on the phenotype of the colonies, progenitors produced in MS-5 cocultures were more mature than those generated on human marrow adherent cells. (2) Hydrocortisone counteracted the stimulatory effect of recombinant human cytokines (interleukin-3, interleukin-6, and steel factor) in assays performed on MS-5 but not on human marrow feeders. (3) Hydrocortisone led to a 50% decrease in the numbers of colony-forming units- granulocyte-macrophage found in methycellulose colony assays of CD34++/CD38- cells performed in the presence of MS-5 cells. Taken together, our results indicate that hydrocortisone acts differently on a murine stromal cell line and on marrow-derived human stromal cells and may suppress the expression by MS-5 cells of an activity selectively promoting amplification of clonogenic cells derived from primitive LTC-IC.

scholarly journals Cloning and Characterization of a Novel Oxidoreductase KDRF from a Human Bone Marrow-derived Stromal Cell Line KM-102

1997 ◽  
Vol 272 (4) ◽  
pp. 2570-2577 ◽  
Author(s):  
Ryuta Koishi ◽  
Ichiro Kawashima ◽  
Chigusa Yoshimura ◽  
Mie Sugawara ◽  
Nobufusa Serizawa
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Stromal Cell Line

scholarly journals Differentiation of early plasma cells on bone marrow stromal cells requires interleukin-6 for escaping from apoptosis

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 487-494 ◽  
Author(s):  
MM Kawano ◽  
K Mihara ◽  
N Huang ◽  
T Tsujimoto ◽  
A Kuramoto
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Interleukin 6 ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Direct Evidence ◽  
Plasma Cells ◽  
Stromal Cell Line ◽  
Culture Supernatants

Abstract The bone marrow (BM) is well known to be the major site of Ig production in secondary immune responses; thus, the microenvironment of BM is considered to be essential for final differentiation of plasma cells. We identified in the peripheral blood (PB) early plasma cells (CD38++CD19+VLA-5-) committed to entering the BM. The sorted early plasma cells rapidly entered apoptosis in vitro, but these cells could survive and further differentiate into mature plasma cells (CD38 CD19+) just as BM plasma cells in the presence of a BM-derived stromal cell line (KM-102). Culture supernatants of KM-102 cell lines could also support survival of these cells, and antibody to interleukin-6 (IL-6) completely blocked the effect of these supernatants. Furthermore, recombinant IL-6, but not IL-1 or IL-3, could support their survival and their differentiation into mature plasma cells (CD38 CD19+VLA-5+) with expression of VLA-5 mRNA. Therefore, here is direct evidence that early plasma cells found in the PB differentiated into mature plasma cells with stromal cell-derived IL-6 in vitro; thus, BM stromal cells control the final checkpoint of plasma cell differentiation with secretion of IL-6 in the BM.

Perfusion Enhances Functions of Bone Marrow Stromal Cells in Three-Dimensional Culture

1998 ◽  
Vol 7 (3) ◽  
pp. 319-326 ◽  
Author(s):  
Julie Glowacki ◽  
Shuichi Mizuno ◽  
Joel S. Greenberger
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Three Dimensional ◽  
Marrow Stromal Cells ◽  
Brdu Incorporation ◽  
Stromal Cell Line ◽  
3D Collagen ◽  
And Function

Perfusion of medium through three-dimensional (3D) collagen sponges enhanced viability and function of cocultivated marrow stromal and hematopoietic cell lines. Cells of the murine bone marrow stromal cell line GPIa were cultured in novel 3D collagen sponges, made from pepsin-digested bovine skin. Static cultures of sponges were maintained in dishes with media changes every other day. Perfused sponges were contained in a glass column with medium flow set at 1.3 mL/min. In some sponges, the 32D cl3 c-fmsm (CRX-1) hematopoietic progenitor cell line was added 7 days after GPIa cells. At 7 and 16 days, light microscopic evaluation showed poor viability of cells in static sponge cultures. In perfused sponge cultures, there was greater cellularity throughout the sponge and abundant accumulation of metachromatic extracellular matrix surrounding GPIa cells. Chondroitin 6-sulfate and heparan sulfate were identified as components of the matrix by immunohistochemical methods. DNA synthesis was evaluated by 15-h exposure of cultures to bromodeoxyuridine (BrdU), with subsequent immunohistochemical localization with monoclonal anti-BrdU antibody. Cells positive for BrdU were identified at the outer surfaces of both static and perfused sponges; however, positive cells were also seen throughout the internal areas of the sponges that were perfused. These results suggest that better nutrient exchange occurred in perfused sponges. In static cocultures of GPIa and CRX-1 cells, there was no detectable viability of the IL-3–dependent CRX-1 cells; however, under perfused conditions, CRX-1 cells flourished within the sponges as documented by BrdU incorporation. Thus, medium perfusion enhanced GPIa stromal cell line viability and function in 3D collagen sponge cultures, as demonstrated by BrdU incorporation, matrix production, and support of CRX-1 cells. This novel culture system may be useful for examining the interactions of bone marrow stromal cells with extracellular matrix molecules, soluble and matrix-bound factors, and with other cell types.

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