scholarly journals Amphotropic retrovirus vector transfer of the v-ras oncogene to human hematopoietic and stromal cells in continuous bone marrow cultures

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 744-752
Author(s):  
L Rothstein ◽  
JH Pierce ◽  
V Klassen ◽  
JS Greenberger
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
High Efficiency ◽  
Leukemia Virus ◽  
Reverse Transcriptase Activity ◽  
Pseudotype Virus ◽  
Bone Marrow Cultures ◽  
Ras P21 ◽  
Nonadherent Cells ◽  
Marrow Cultures

Human continuous bone marrow cultures were established from intraoperative marrow specimens and infected with amphotropic murine leukemia virus (Am-MuLV) pseudotypes of Kirsten or Harvey murine sarcoma virus, and the biologic effects were compared with mouse continuous bone marrow cultures. Cultures were tested for production of total nonadherent granulocytes and granulocyte-macrophage progenitor cells (GM-CFUc); virus replication by supernatant reverse transcriptase activity; percentage of adherent and nonadherent cells and GM-CFUc that released virus by infectious center assay; and for synthesis of Harvey ras p21 protein. High-efficiency, stable Am-MuLV infection of over 90% of human marrow-culture nonadherent and adherent cells and both seven- and 14-day GM-CFUc were detected as Kirsten or Harvey pseudotype virus release by infectious center assay. Synthesis of Harvey ras p21 was detected in the adherent and nonadherent cell populations of human as well as mouse continuous marrow cultures infected with Kirsten or Harvey pseudotype virus. In contrast to data with mouse cultures, cumulative production of GM-CFUc and differentiated granulocytes in human cultures was not detectably altered by Harvey or Kirsten virus infection, and all cultures ceased to produce hematopoietic cells by 20 weeks. Of 54 virus-infected cultures in ten separate experiments, 13 produced a second peak of nonadherent cells (greater than 10(5) per flask) after 20 weeks, significantly more frequently than did control uninfected cultures (one of 32). When subcultured, these harvests produced permanent Epstein-Barr virus (EBV)-transformed pre-B cell lines that released the original inoculating pseudotype virus. Thus, Am- MuLV is a potentially valuable vector for inserting genetic sequences by recombinant techniques into human hematopoietic and stromal cells in culture; however, activation of EBV may be a significant complication.

scholarly journals Amphotropic retrovirus vector transfer of the v-ras oncogene to human hematopoietic and stromal cells in continuous bone marrow cultures

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 744-752 ◽  
Author(s):  
L Rothstein ◽  
JH Pierce ◽  
V Klassen ◽  
JS Greenberger
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
High Efficiency ◽  
Leukemia Virus ◽  
Reverse Transcriptase Activity ◽  
Pseudotype Virus ◽  
Bone Marrow Cultures ◽  
Ras P21 ◽  
Nonadherent Cells ◽  
Marrow Cultures

Abstract Human continuous bone marrow cultures were established from intraoperative marrow specimens and infected with amphotropic murine leukemia virus (Am-MuLV) pseudotypes of Kirsten or Harvey murine sarcoma virus, and the biologic effects were compared with mouse continuous bone marrow cultures. Cultures were tested for production of total nonadherent granulocytes and granulocyte-macrophage progenitor cells (GM-CFUc); virus replication by supernatant reverse transcriptase activity; percentage of adherent and nonadherent cells and GM-CFUc that released virus by infectious center assay; and for synthesis of Harvey ras p21 protein. High-efficiency, stable Am-MuLV infection of over 90% of human marrow-culture nonadherent and adherent cells and both seven- and 14-day GM-CFUc were detected as Kirsten or Harvey pseudotype virus release by infectious center assay. Synthesis of Harvey ras p21 was detected in the adherent and nonadherent cell populations of human as well as mouse continuous marrow cultures infected with Kirsten or Harvey pseudotype virus. In contrast to data with mouse cultures, cumulative production of GM-CFUc and differentiated granulocytes in human cultures was not detectably altered by Harvey or Kirsten virus infection, and all cultures ceased to produce hematopoietic cells by 20 weeks. Of 54 virus-infected cultures in ten separate experiments, 13 produced a second peak of nonadherent cells (greater than 10(5) per flask) after 20 weeks, significantly more frequently than did control uninfected cultures (one of 32). When subcultured, these harvests produced permanent Epstein-Barr virus (EBV)-transformed pre-B cell lines that released the original inoculating pseudotype virus. Thus, Am- MuLV is a potentially valuable vector for inserting genetic sequences by recombinant techniques into human hematopoietic and stromal cells in culture; however, activation of EBV may be a significant complication.

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

1986 ◽  
Vol 163 (4) ◽  
pp. 872-883 ◽  
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.

scholarly journals 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 ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
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.

Increased longevity of hematopoiesis in continuous bone marrow cultures and adipocytogenesis in marrow stromal cells derived from Smad3−/− mice

2005 ◽  
Vol 33 (3) ◽  
pp. 353-362 ◽  
Author(s):  
Michael W. Epperly ◽  
Shaonan Cao ◽  
Julie Goff ◽  
Donna Shields ◽  
Shuanhu Zhou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Marrow Stromal Cells ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

scholarly journals 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 ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
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

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.

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

Blood ◽  
1984 ◽  
Vol 64 (2) ◽  
pp. 516-525 ◽  
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.

scholarly journals Role of colony-stimulating activity in murine long-term bone marrow cultures: evidence for its production and consumption by the adherent cells

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 761-767 ◽  
Author(s):  
JM Heard ◽  
S Fichelson ◽  
B Varet
Keyword(s):  
Bone Marrow ◽  
Myeloid Cells ◽  
Target Cells ◽  
Adherent Cells ◽  
Colony Stimulating Activity ◽  
Large Numbers ◽  
Bone Marrow Cultures ◽  
Nonadherent Cells ◽  
Marrow Cultures

Abstract The involvement of colony-stimulating activity (CSA) in murine long- term bone marrow cultures (LTBMC) was studied using bilayer agar cultures. The supernatants of LTBMC were removed, a layer of dense agar was spread over the cells adherent to the bottom of the flask, and fresh myeloid cells were plated as source of CFU-C in an upper agar layer. Large numbers of granulocytic and macrophagic colonies developed regularly when target cells were plated over adherent cells of nonrecharged and greater than 12 wk old LTBMC that were hematopoietically inactive (i.e., producing a low number of nonadherent cells). The removal of adherent cells from the myeloid cells used as source of CFU-C did not decrease the number of colonies. This suggests that adherent cells of LTBMC release CSA that is directly active on CFU- C. This CSA was no longer detectable over adherent layers of hematopoietically active LTBMC. A close inverse relationship was demonstrated between the number of nonadherent cells harvested before the assay and the level of CSA. No inhibitor for CSA was demonstrated in the supernatant of hematopoietically active cultures. Murine exogenous CSA incubated over the adherent layer host its activity within 24 hr, whereas in the same conditions human CSA retained its activity. These data demonstrate the production of CSA by the adherent layer of LTBMC and strongly suggest its specific in situ consumption by differentiating myeloid cells.

Sign in / Sign up

Export Citation Format

Share Document