Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.     

Radiosensitivity of colony-forming units of dog bone marrow in agar cultures]

Radiosensitivity of the bone-marrow colony-forming cells was determined by a modified method of hemopoietic cells cloning in vivo in semihard agar gel in diffusion chambers. Do for the commited precursor cells of granulopoiesis (CFUc) was 144 +/- 14.8 rad (n = 0.8), and Do for the precursor cells forming "stellate" colonies of fibroblast-like cells (PFUf) was 468 +/- 35.8 rad (n == 0.9). A conclusion was drawn that PFUf were referred to the class of stromal precursors of the hemopoietic organs. This system can be applied for a simultaneous study of the hemopoietic and stromal precursor cells in dogs.     

Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells

We have systematically studied the effect on hemopoietic colony-forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30-60 min incubation with a 5 microM concentration of stain. Flow-cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony-forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU-M, a megakaryocyte colony-forming cell; CFU-E, a relatively differentiated erythroid precursor; BFU-E, a primitive erythroid precursor; CFU-GM, a granulocyte-macrophage precursor; and CFU-S, the spleen colony-forming cell or hemopoietic stem cell. A staining procedure involving a 30-min exposure to 5 microM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU-M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.     

Insertion of the bacterial gene for dihydrofolate reductase into colony-forming cells of mouse bone marrow

Introduction of the plasmid containing the methotrexate-resistant (Mtx-r) bacterial gene of dihydrofolate reductase (DHFR) under the control of the early promoter of SV 40 into the donor bone cells of the mouse with subsequent transplantation of the cells into lethally irradiated mice results in the increase in the life span of mice under conditions of methotrexate selection. It is due to the stable transformation of the bone marrow colony-forming cells with the plasmic DNA and the synthesis of the bacterial Mtx-r DHFR in the spleen and bone marrow of the recipient mouse.     

Proliferative and differentiation potentials of skeletogenic bone marrow colony-forming cells

The clonal nature of bone marrow fibroblast colonies derived from clonogenic bone marrow osteogenic cells (CFUf) was proved by the chromosome analysis. During subsequent passages of multi-colony derived bone marrow fibroblast strains there occurs a pronounced increase in the cell number and in the number of osteogenic units (tested by transplantation in diffusion chambers). Single colony-derived strains are capable of forming bone and cartilage simultaneously. It follows that CFUf or part of them are clonogenic cells with high proliferative potentials and are common precursors for bone and cartilage tissue. Thus, CFUf may be regarded as osteogenic stem cells.     

Characteristics of proliferation and differentiation of spleen colony-forming cells from bone marrow

By using c-band staining or sex chromosome identification techniques, we have demonstrated that some of the spleen colony-forming cells from normal bone marrow have the potential to form both myeloid and lymphoid elements.     

Radiosensitivity of mouse bone marrow and spleen CFUdc during gamma-irradiation in different oxygenation conditions

After exposure in vitro and in situ CFUdc of CBA mouse bone marrow and spleen were characterized by the comparable parameters of radiosensitivity and oxygen-dependent modification: the values of D0 for bone marrow and spleen cells were 1.31 and 1.35 Gy (in vitro) and 1.36 and 1.37 Gy (in situ), and the values of the oxygen effect were 2.3 and 2.5, respectively.     

Erythroid cells of the bone marrow of the mouse

The bone marrow of three intact male mice of C57Bl/6 line, fixed by perfusion of isotonic fixative of Karnovsky, has been studied by means of the scanning electron microscopy method. The surface of erythroid cells, that are immediately connected with macrophages of the erythroblasts islets, is analysed. According to the surface form, the erythroid cells are devided into 5 types. Every maturation stage of the erythroid cells is characterized by a certain type of surface. For identification of basophilic and polychromatophilic proerythrocytes the combined method of light and electron scanning microscopy of the cells in suspension is used. The bone marrow cells, obtained from the two male mice of C57Bl/6 line are fixed with the same fixative on special glasses with grids traced on them, stained after Romanovsky-Giemsa method and in moist preparations are examined in the light microscope. After further treatment the surface of the same cells in studied in the scanning electron microscope.     

Reaction of bone marrow colony-forming cells in irradiated mice to phlogogenic factors]

The experiments on irradiated mice (600 and 800 r) demonstrated that under the influence of flogogenic factors the quantity of colonies in the spleen increases and their morphological structures change. It is suggested that under the action of the damaging agent the "hypothetic factor" which causes the pointed changes, is formed in the skin.     

Immunoglobulin-positive cells in mouse bone marrow]

The content of Ig-bearing lymphocytes and their precursors in the mouse bone marrow was investigated 6 and 36 hours after the hydroxyurea treatment. Some increase of the B-cell content takes place in the trated bone marrow. Dividing and non-dividing B-cell precursors, except the stem cells, were practically absent.     

Effect of synthetic polyribonucleotides on the immunological and colony-forming activity of irradiated bone marrow cells

The experimental data are presented concerning the effect of polyribonucleotides on the immunologic and colony forming ability of bone marrow or irradiated mice. All the compounds under study exhibited a pronounced, but to a different degree, colony-forming and immunostimulating action. The comparative study of the influence of polyribonucleotides on the number of endogenous colonies and antibody-forming cells showed an inverse relationship between these parameters: The preparations exerting the most pronounced immunostimulating effect had an insignificant colony-forming action and vice versa. This is evidently indicative of the capacity of these preparations to turn the differentiation of haemopoietic stem cells towards the immunopoiesis.     

Detection of in vitro macrophage colony-forming cells (M-CFC) in mouse bone marrow,spleen, and peripheral blood

In vitro macrophage colony-forming cells (M-CFC) have been detected in bone marrow (BM) (317/10⁵ cells), spleen (SPL) (81/10⁵), and peripheral blood leukocytes (PBL) (242/10⁵) of the mouse. These M-CFCs were similar to those previously detected in thymus (T) (30/10⁶) and lymph node (LN) (22/10⁶) tissue in several respects. BM- and SPL-derived M-CFC required PMUE to consistently initiate colony formation, whereas PBL-derived M-CFC formed colonies with stimulation by either PMUE or L-cell-conditioned medium. All colonies formed showed a singular macrophage line of differentiation, a lag of 13 to 18 days prior to initiating colony formation, a marked ability to survive in culture in the absence of PMUE, and markedly slow rates of appearance in culture once colony formation was initiated. The macrophage progeny were identified on the basis of morphology, glass adherence, the phagocytosis of agar, bacteria and SRBC, and the presence of receptors for IgG. These characteristics are also shared by those macrophage CFCs observed within stimulated peritoneal exudate, pleural effusion, and alveolar space. These M-CFCs are most likely members of a large, heterogeneous population of macrophage progenitor cells distributed throughout the hemato-lymphopoietic organs, serosal cavities and surfaces, and inflammatory and alveolar tissue sites. The degree of heterogeneity may be determined in part by the influence of tissue-specific microenvironment.