Irradiated Stroma Research Articles

Differentiation of primitive human multipotent hematopoietic progenitors into single lineage clonogenic progenitors is accompanied by alterations in their interaction with fibronectin.

We have previously demonstrated that primitive progenitors from human bone marrow termed long term bone marrow culture initiating cells (LTBMC-IC) adhere avidly to irradiated bone marrow stroma, while more mature clonogenic progenitors fail to do so. In this study we examine the interaction between these progenitors and components of the bone marrow stroma. (a) We demonstrate that both primitive LTBMC-IC and more mature clonogenic progenitors adhere to intact fibronectin. (b) Primitive LTBMC-IC and multi-lineage CFU-MIX progenitors adhere to the 33/66 kD COOH-terminal heparin-binding cell-adhesion promoting fragment of fibronectin, but adhere significantly less to its 75 kD RGDS-dependent cell-binding fragment. In contrast, more differentiated single-lineage progenitors adhere equally well to the 33/66 kD RGDS independent and the 75 kD RGDS-dependent cell-adhesion fragments of fibronectin. (c) Both primitive LTBMC-IC and clonogenic progenitors adhere to the three known cell-attachment sites in the 33/66 kD cell-adhesion promoting fragment, FN-C/H I, FN-C/H II and CS1. However, LTBMC-IC and CFU-MIX progenitors adhere significantly better to FN-C/H II than to the flanking FN-C/H I and CS1 cell-attachment sites. In contrast, single-lineage progenitors adhere equally well to all three cell attachment sites in the 33/66 kD cell-adhesion promoting fragment. (d) Finally, adhesion of primitive LTBMC-IC to intact irradiated stroma can be inhibited partially by peptide FN-C/H II and almost completely by a combination of FN-C/H II and peptide FN-C/H I and CS1. This study demonstrates that adhesive interactions between primitive hematopoietic progenitors and the extracellular matrix component fibronectin can occur. Specific changes in adhesion to the 33/66 kD cell-adhesion promoting fragment and the 75 kD RGDS-dependent cell-adhesion fragment of fibronectin are associated with differentiation of primitive multi-lineage progenitors into committed single-lineage progenitors. Such differences in adhesive interaction with fibronectin may allow hematopoietic progenitors at various stages of differentiation to interact with specific supportive loci of the bone marrow microenvironment. Finally, the ability to block adhesion of LTBMC-IC to intact irradiated stroma with peptides FN-C/H II, FN-C/H I and CS1 suggests that receptors responsible for this interaction may be important in the homing of primitive progenitors to the bone marrow.

Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma.

We enriched bone marrow cells from 10 normal individuals for primitive hematopoietic progenitors using a two-step technique, and examined resultant primitive progenitors for their in vitro long-term repopulating capacity and their ability to adhere to irradiated stroma. Immunomagnetic depletion of mature myeloid and lymphoid progenitors resulted in a lineage-negative (Lin-) cell population. Subsequent dual-color fluorescence activated sorting of cells with low forward and vertical light scatter properties, expressing CD34 antigen (34+) and either bearing (DR+) or lacking (DR-) the HLA-DR antigen, resulted in the selection of Lin-34+ DR+ and Lin-34+ DR- cell populations. When the Lin-34+ DR+ cell fraction was cultured in a short-term methylcellulose assay, we demonstrated a 61-fold enrichment for colony forming cells (CFC) compared with undepleted bone marrow mononuclear cells. In contrast to the Lin-34+ DR+ cells, direct culture of Lin-34+ DR- cells in short-term methylcellulose generated significantly less CFC (p less than or equal to 0.001). We then compared the capacity of Lin-34+ DR+ and Lin-34+ DR- cells to generate sustained hematopoiesis when plated in long-term bone marrow culture (LTBMC). When LTBMC were initiated with plated Lin-34+ DR+ cells, we recovered high numbers of CFC during the first week, but observed a rapid decline in the number of harvested CFC over the following weeks. No CFC could be recovered after week 7. In contrast, LTBMC initiated with plated Lin-34+ DR- cells yielded significantly greater numbers of CFC than LTBMC initiated with plated Lin-34+ DR+ cells (p less than or equal to 0.001), and this was sustained for at least 12 wk of culture. The Lin-34+ DR+ population was only 6.6-fold enriched for primitive progenitors capable of initiating and sustaining hematopoiesis in LTBMC when compared with undepleted bone marrow mononuclear cells, while the Lin-34+ DR- population was 424-fold enriched for such primitive progenitors (p less than or equal to 0.001). Finally, we examined the capacity of both Lin-34+ DR+ and Lin-34+ DR- populations to adhere to irradiated allogeneic stroma. We used a previously described "panning method" in which cells are plated onto stroma for 2 h, the nonadherent cells removed by extensive washing, and the adherent fraction maintained under conditions favoring LTBMC growth. When stroma was panned with Lin -34+ DR+ cells, 79 +/- 10% of the cells were recovered in the panning effluent. In contrast, when stroma was panned with Lin -34 + DR- cells, significantly fewer (37 +/- 7%) (p less than or equal to 0.001) cells were recovered in the panning effluent. Unlike LTBMC initiated with plated Lin -34 + DR+ cells, virtually no CFC were recovered from LTBMC initiated with panned Lin -34 + DR+ cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Tumor bed expression in xenografted artificial heterogeneous colon tumors

Artificial heterogeneous xenograft tumors (AHTs) were produced by injecting admixtures of two clonal cell lines (A and D) originally isolated from the DLD-1 human colon adenocarcinoma. The volumetric growth of unirradiated pure A or D tumors and 2 different admixtures (approximately 90% A + 10% D or 10% A + 90% D) were compared to growth of similar tumors growing in normal tissue stroma that had received 5, 10, or 15 Gy of X rays 1 day prior to cell injections. The X-irradiations produced a dose dependent delay in growth at sizes of 500 mm 3 (designated as the tumor bed effect, TBE) for both pure and admixed tumors of about 2 at 15 Gy. There were no significant differences in TBE among the various groups. Also, determination of tumor cell yields, or colony forming efficiencies, showed no differences among tumors growing in undamaged or damaged stroma. However, beginning at about 20 days postirradiation, there was a significant change in the percentage composition of the tumors growing in the irradiated stroma. Both admixtures became significantly enriched in the subpopulation that was originally predominant. In contrast, the percentage compositions of the admixtures growing in unirradiated stroma remained stable over the duration of the experiment period (to about 70 days postirradiation). We interpret these data to indicate that the radiation damage to the normal tissue produced an intratumor environment leading to competitive exclusion dynamics of the minority subpopulation. Clinical implications of these results are discussed.

Stromal growth factor production in irradiated lectin exposed long-term murine 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.

Stromal Growth Factor Production in Irradiated Lectin Exposed Long-Term Murine 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.

Tumour Bed Effect: Hypoxic Fraction of Tumours Growing in Preirradiated Beds

The reduction in tumour growth rate seen when tumours are implanted into preirradiated sites, the tumour bed effect (TBE), is believed to be due to radiation damage to vascular stroma, leading to defective angiogenesis in the tumour. The present work examined whether or not the functional inadequacy of irradiated stroma was accompanied by an increased hypoxic fraction in tumours growing in irradiated beds. Mouse flank skin was given 0 or 20 Gy X-rays and RIF-1 fibrosarcoma cells were implanted i.d. into the centre of the treatment field one week later. Tumours of 200 mm3 were irradiated under clamped or unclamped conditions and the hypoxic fraction measured from the displacement of the corresponding survival curves, assayed in vitro. Results indicated a small increase in the hypoxic fraction. Averaging values from three independent experiments, the percentage of hypoxic cells increased from 2.5 per cent for cells in tumours growing in unirradiated beds to 4.6 per cent for those from tumours in beds given 20 Gy. Thus an irradiated vascular bed is still to some extent able to maintain the proportion of oxic: hypoxic tumour cells found in tumours growing in unirradiated beds, despite manifest changes in tumour necrosis and growth rate.

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

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.

Hematopoietic Regulatory Factors Produced in Long-Term Murine Bone Marrow Cultures and the Effect of In Vitro Irradiation

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.

Lithium stimulation of murine hematopoiesis in liquid culture: an effect mediated by marrow stromal cells

Lithium has previously been observed to stimulate in vitro Dexter culture hemopoiesis with increases in granulocytes, megakaryocytes, and pluripotent stem cells (CFU-S). In the present study, a two-phase murine Dexter culture system was established to study the mechanism of lithium-mediated stem cell stimulation. Different lots of horse sera or fetal calf sera were found to have markedly different effects on Dexter culture growth; given the appropriate sera supplementation, supernatant cells from Dexter cultures established from C57BL/6J mice 3 wk previously were free of stromal-forming capacity, but had stem cells and could grow on 900–950 R irradiated stroma. Conversely, in vitro irradiation (900–950 R) of 3-wk cultures resulted in a stem-cell-free adherent monolayer that could support growth for up to 9 wk in culture. The stroma from Dexter cultures preexposed to lithium chloride (1.0 mmole/liter) for 3 wk, irradiated (900 R), and then refed with 3-wk Dexter supernatant cells has an enhanced capacity to support cell production, CFU-S, and probably granulocyte-macrophage colony-forming cell (GM-CFU-C) production, as compared to stroma not preexposed to lithium. Lithium carryover was ruled out in these experiments. These data indicate that lithium stimulates CFU-S and in vitro granulopoiesis by an indirect effect on a radioresistant adherent stromal cell.

Lithium Stimulation of Murine Hematopoiesis in Liquid Culture: An Effect Mediated by Marrow Stromal Cells

Lithium has previously been observed to stimulate in vitro Dexter culture hemopoiesis with increases in granulocytes, megakaryocytes, and pluripotent stem cells (CFU-S). In the present study, a two-phase murine Dexter culture system was established to study the mechanism of lithium-mediated stem cell stimulation. Different lots of horse sera or fetal calf sera were found to have markedly different effects on Dexter culture growth; given the appropriate sera supplementation, supernatant cells from Dexter cultures established from C57BL/6J mice 3 wk previously were free of stromal-forming capacity, but had stem cells and could grow on 900-950 R irradiated stroma. Conversely, in vitro irradiation (900-950 R) of 3-wk cultures resulted in a stem-cell-free adherent monolayer that could support growth for up to 9 wk in culture. The stroma from Dexter cultures preexposed to lithium chloride (1.0 mmole/liter) for 3 wk, irradiated (900 R), and then refed with 3-wk Dexter supernatant cells has an enhanced capacity to support cell production, CFU-S, and probably granulocyte-macrophage colony-forming cell (GM-CFU-C) production, as compared to stroma not preexposed to lithium. Lithium carryover was ruled out in these experiments. These data indicate that lithium stimulates CFU-S and in vitro granulopoiesis by an indirect effect on a radioresistant adherent stromal cell.

Stimulation and support of haemopoietic stem cell proliferation by irradiated stroma cell colonies in bone marrow cell culture in vitro.

A culture system was established in which haemopoietic stem cells can undergo a recovery proliferation after a depletion of the stem cells, completely in vitro. To elucidate the source of the stimulatory factors, normal bone marrow cells were overlayed on top of the irradiated adherent ‘stromal’ cell colonies in the bone marrow cell culture. This stimulated the proliferation of haemopoietic stem cells in the cultured cells in suspension. The present results indicate that the stromal cells produce factors which stimulate stem cell proliferation. Whether the stimulation is evoked by direct cell-cell interactions or by humoral factors is as yet to be studied.