Low level of gene transfer to and engraftment of murine bone marrow cells from long-term bone marrow cultures

Hematopoietic regulatory factors produced by adherent (stromal) cells in long-term murine bone marrow cultures have been investigated. Using an in situ double layer agar overlay system, we demonstrated that exposure of the stromal cells to 1,100-rad irradiation increased their activities in stimulating colony formation of FDC-P1, an interleukin 3 (IL 3)-responsive cell line. The colony-stimulating activities (CSAs) of the irradiated stroma also stimulated normal marrow cells to form granulocyte-macrophage, megakaryocyte, and mixed lineage colonies. Addition of the lectin pokeweed mitogen to the irradiated stroma increased the level of CSAs. The FDC-P1 CSA of the irradiated stroma was inhibited by antibodies directed against murine granulocyte- macrophage colony stimulating factor (GM-CSF) but not by those against murine IL 3. Stromal-derived CSA for marrow cells was also partially blocked by anti-GM-CSF antibodies, probably reflecting the presence of other CSAs such as CSF-1. This latter growth factor has been found to be present in conditioned media from Dexter stroma, but levels are not increased after irradiation or lectin exposure. Partially purified GM- CSF, like IL 3, stimulated FDC-P1 proliferation and granulocyte, macrophage, and megakaryocyte colony formation. These results indicate that the major terminal differentiating hormone elicited by irradiation or lectin exposure of murine marrow stromal cells is GM-CSF. This growth factor, along with CSF-1, can account for the differentiated progeny produced in this system: macrophages, granulocytes, and megakaryocytes.

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Stromal growth factor production in irradiated lectin exposed long-term murine bone marrow cultures

Blood ◽

10.1182/blood.v69.4.1120.1120 ◽

1987 ◽

Vol 69 (4) ◽

pp. 1120-1127 ◽

Cited By ~ 20

Author(s):

TA Alberico ◽

JN Ihle ◽

CM Liang ◽

HE McGrath ◽

PJ Quesenberry

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Stromal Cells ◽

Colony Formation ◽

Murine Bone Marrow ◽

Gm Csf ◽

Bone Marrow Cultures ◽

Marrow Cultures ◽

Marrow Cells

Abstract Hematopoietic regulatory factors produced by adherent (stromal) cells in long-term murine bone marrow cultures have been investigated. Using an in situ double layer agar overlay system, we demonstrated that exposure of the stromal cells to 1,100-rad irradiation increased their activities in stimulating colony formation of FDC-P1, an interleukin 3 (IL 3)-responsive cell line. The colony-stimulating activities (CSAs) of the irradiated stroma also stimulated normal marrow cells to form granulocyte-macrophage, megakaryocyte, and mixed lineage colonies. Addition of the lectin pokeweed mitogen to the irradiated stroma increased the level of CSAs. The FDC-P1 CSA of the irradiated stroma was inhibited by antibodies directed against murine granulocyte- macrophage colony stimulating factor (GM-CSF) but not by those against murine IL 3. Stromal-derived CSA for marrow cells was also partially blocked by anti-GM-CSF antibodies, probably reflecting the presence of other CSAs such as CSF-1. This latter growth factor has been found to be present in conditioned media from Dexter stroma, but levels are not increased after irradiation or lectin exposure. Partially purified GM- CSF, like IL 3, stimulated FDC-P1 proliferation and granulocyte, macrophage, and megakaryocyte colony formation. These results indicate that the major terminal differentiating hormone elicited by irradiation or lectin exposure of murine marrow stromal cells is GM-CSF. This growth factor, along with CSF-1, can account for the differentiated progeny produced in this system: macrophages, granulocytes, and megakaryocytes.

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Differentiation of dendritic cells in cultures of rat bone marrow cells.

Journal of Experimental Medicine ◽

10.1084/jem.163.4.872 ◽

1986 ◽

Vol 163 (4) ◽

pp. 872-883 ◽

Cited By ~ 45

Author(s):

W E Bowers ◽

M R Berkowitz

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Bone Marrow Cells ◽

Sensitive Assay ◽

Ia Antigens ◽

Bone Marrow Cultures ◽

Nonadherent Cells ◽

Marrow Cultures ◽

Rat Bone ◽

Marrow Cells

Although dendritic cells (DC) originate from bone marrow, they were not observed in fresh preparations of bone marrow cells (BMC). Likewise, accessory activity was barely measurable in a sensitive assay for this potent function of DC. However, both DC and accessory activity developed when BMC were cultured for 5 d. Based on fractionation before culture, nearly all of the accessory activity could be attributed to only 5% of the total BMC recovered in a low-density (LD) fraction. The LD-DC precursors differed from mature DC in a number of important respects. Removal of Ia+ cells from the LD fraction by panning did not decrease the production of DC when the nonadherent cells were cultured. Thus, the cell from which the DC is derived does not express or minimally expresses Ia antigens, in contrast to the strongly Ia+ DC that is produced in bone marrow cultures. Irradiation of LD cells before culture prevented the development of DC. When irradiation was delayed by daily intervals, progressive increases in the number of DC resulted, up to the fifth day. These findings, together with preliminary autoradiographic data, indicate that cell division has occurred, in contrast to the DC, which does not divide. We conclude that bone marrow-derived DC arise in culture from the division of LD, Ia- precursors.

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Hematopoietic regulatory factors produced in long-term murine bone marrow cultures and the effect of in vitro irradiation

Blood ◽

10.1182/blood.v64.2.516.bloodjournal642516 ◽

1984 ◽

Vol 64 (2) ◽

pp. 516-525 ◽

Cited By ~ 1

Author(s):

RJ Gualtieri ◽

RK Shadduck ◽

DG Baker ◽

PJ Quesenberry

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Factor Vii ◽

Murine Bone Marrow ◽

L Cell ◽

Bone Marrow Cultures ◽

Macrophage Colony ◽

Marrow Cultures

The nature of hematopoietic regulatory factors elaborated by the adherent (stromal) cells of long-term murine bone marrow cultures and the effect of in vitro stromal irradiation (XRT) on the production of these factors was investigated. Using an in situ stromal assay employing a double layer of semisolid agar, it was possible to demonstrate stromal elaboration of at least two colony-stimulating activities, ie, granulocyte/macrophage colony-stimulating activity (G/M- CSA) and megakaryocyte colony-stimulating activity (Meg-CSA). Exposure of the stroma to XRT resulted in dose-dependent elevations of both activities that correlated inversely with total myeloid cell mass as determined by concurrent reductions in total supernatant cell recoveries from irradiated cultures. Mixture experiments that combined control and irradiated stroma revealed that the hematopoietically active control stroma could block detection of XRT-related G/M-CSA elevations. These data implicate a local negative feedback mechanism in the regulation of hematopoiesis. Antiserum directed against purified L cell colony-stimulating factor (CSF) reduced granulocyte/macrophage colony formation in the target layer but did not effect the increased Meg-CSA. While a radioimmunoassay for L-cell type CSF was unable to detect significant differences in concentrated media from control and irradiated cultures, bioassays of these media revealed XRT-related G/M- CSA elevations. These results indicate that the G/M-CSA elaborated in these cultures is immunologically distinct from the Meg-CSA produced, and although distinct from L cell CSF, the G/M-CSA is crossreactive with the L cell CSF antiserum. Morphologic, histochemical, and factor VII antigen immunofluorescent studies were performed on the stromal cell population responsible for production of these stimulatory activities. In addition to “fat” cells, the stromal cells remaining after XRT were composed of two predominant cell populations. These included a major population of acid phosphatase and nonspecific esterase-positive macrophage-like cells and a minor population of factor VII antigen negative epithelioid cells.

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No stimulative effect of adipocytes on hematopoiesis in long-term human bone marrow cultures

Blood ◽

10.1182/blood.v61.4.770.bloodjournal614770 ◽

1983 ◽

Vol 61 (4) ◽

pp. 770-774

Author(s):

I Touw ◽

B Lowenberg

Keyword(s):

Bone Marrow ◽

Human Bone ◽

Human Bone Marrow ◽

Adherent Cell ◽

Murine Bone Marrow ◽

Bone Marrow Cultures ◽

Human Bone Marrow Cultures ◽

Marrow Cultures

Long-term cultures of human bone marrow were established for 5–13 wk to study the role of adipocytes in sustaining hematopoiesis. At weekly intervals, the numbers of nucleated cells and granulocyte-macrophage progenitor cells (GM-CFU) in culture were estimated in relation to the numbers of fat-containing cells present in the adherent stroma layer. In these quantifications, the numbers of GM-CFU trapped in the adherent cell layer were considered separately. It was found that the presence of adipocytes did not correlate with more active hematopoiesis. Fat cells appeared at late stages when successful cultures were being exhausted or early in cultures with poor activity. These observations suggest that human marrow continuous hematopoiesis in vitro, unlike hematopoiesis in the analogous murine bone marrow cultures, does not depend on the presence of adipocytes.

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Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression

Blood ◽

10.1182/blood.v78.2.310.bloodjournal782310 ◽

1991 ◽

Vol 78 (2) ◽

pp. 310-317 ◽

Cited By ~ 1

Author(s):

JF Apperley ◽

BD Luskey ◽

DA Williams

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Bone Marrow Cells ◽

Selectable Marker ◽

Retroviral Vector ◽

G418 Selection ◽

Marrow Cells

Retroviral-mediated gene transfer of human adenosine deaminase (hADA) provides a model system for the development of somatic gene therapy as a therapy for diseases of bone marrow-derived cells. We have previously demonstrated that hADA can be observed in all hematopoietic lineages in a minority of mice transplanted with bone marrow cells infected with a simplified retroviral vector, ZipPGK-ADA. Here we report a majority of mice (six of eight) demonstrate expression of hADA in the peripheral blood at least 6 months after transplantation with bone marrow infected with this simplified retroviral vector, which contains no selectable marker. The failure to express hADA in two of eight mice was associated with the absence of the recombinant retroviral provirus in DNA prepared from bone marrow cells of these mice apparently due to failure to efficiently infect the reconstituting hematopoietic stem cell. In an effort to preselect bone marrow stem cells containing proviral integrations, we incorporated the selectable marker neo phosphotransferase (NEO) into a retroviral vector encoding hADA, N2/ZipPGK-ADATKNEO, and used G418 selection of infected bone marrow cells before transplantation. In contrast to the simplified retroviral vector, hADA expression in these recipients was short lived (less than 8 weeks), despite the continued presence of intact provirus in DNA prepared from bone marrow of these mice. To determine whether the preselection of bone marrow using G418 was responsible for the lack of sustained hADA expression, we repeated the infection with the N2/ZipPGK- ADATKNEO vector but omitted the G418 selection step. Again, the majority of recipient mice failed to express hADA long term, although the continued presence of provirus in DNA prepared from peripheral blood cell mononuclear cells was clearly demonstrated. Finally, we demonstrate clonal fluctuation of infected stem cells, and observe a temporal correlation between cessation of expression of hADA and the emergence of a dominant stem cell clone between 14 and 20 weeks posttransplantation in one recipient. These data suggest that inclusion of a second transcriptional unit that includes neo phosphotransferase sequences in this simplified vector is associated with decreased expression of the nonselectable ADA sequences.

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Extracellular matrix production by the adherent cells of long-term murine bone marrow cultures

Blood ◽

10.1182/blood.v61.3.540.540 ◽

1983 ◽

Vol 61 (3) ◽

pp. 540-547 ◽

Cited By ~ 110

Author(s):

KS Zuckerman ◽

MS Wicha

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Stromal Cells ◽

Extracellular Matrix Proteins ◽

Matrix Proteins ◽

Immunofluorescent Staining ◽

Murine Bone Marrow ◽

Bone Marrow Cultures ◽

Marrow Cultures

Abstract We have studied the deposition of extracellular matrix proteins in the adherent stroma of long-term murine bone marrow cultures. Stable hematopoiesis was maintained for greater than 12 wk. At selected intervals, culture dishes were sacrificed by removing all nonadherent cells and air drying the dishes. The adherent stromal layer was analyzed for the presence of intracellular and extracellular collagen, fibronectin, and laminin using double immunofluorescent staining with specific antisera against these matrix components. In cultures examined during the first 2 wk, large numbers of stromal cells contained collagen, fibronectin, and laminin. Over the next 2 wk, an extensive extracellular network of fibronectin, laminin, and collagen was deposited on the dishes, which persisted throughout the life of the cultures. In contrast to a previous report, we detected substantial numbers of endothelial cells by means of immunofluorescent staining of stromal cells with antisera to type IV collagen, laminin, and factor VIII antigen. Although deposition of these extracellular matrix proteins coincides with onset of active hematopoietic cell production, the relative roles of the stromal cells and the extracellular matrix in supporting hematopoiesis in murine bone marrow cell cultures remain to be determined.

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