Normal Rat Bone Marrow Research Articles

In vitro growth inhibition of Dunning rat prostate tumor by bone marrow factor.

Normal Copenhagen rat bone marrow was assayed for growth inhibition of cultured MAT LyLu rat prostate tumor cells. A marrow-derived factor was identified that had significant growth inhibitory activity in vitro against MAT LyLu as well as against DU-145 human prostate tumor and MBT-2 mouse bladder tumor cells but that was noninhibitory to normal rat fibroblasts. The factor was stable to degradation by acid, heat, freezing, trypsin, and carboxypeptidase B. The factor was nonreactive with Coomassie blue, and the molecular weight was estimated as less than 620 daltons. A similar factor was identified in normal human and normal rat sera. The presence of this factor in bone marrow may explain the absence of osseous metastases in the Dunning rat prostate tumor model.

Pluripotent hemopoietic stem cells give rise to osteoclasts

Osteopetrosis in the ia (incisors absent) rat is the result of reduced bone resorption due to abnormal osteoclasts. The mutant osteoclasts lack a ruffled border--the membrane specialization involved in osteolysis. Studies in the ia mutant have shown that when pluripotent hemopoietic stem cells from normal littermates are transplanted into ia recipients, normal osteoclasts are formed and the skeletal sclerosis is eventually cured. The present study was conducted to provide evidence for the mechanism of the cure. Do the transplanted stem cells provide a helper function, i.e. secrete soluble factor(s) which transform pre-existing osteoclasts, or do they fuse with each other or pre-existing osteoclasts, or do they fuse with each other or pre-existing osteoclasts to form functional osteoclasts? Using the procedures described by Gold-schneider and co-workers, and fluorescence-activated cell sorting (FACS), pluripotent hemopoietic stem cells were isolated from normal rat bone marrow, labeled with saturated FITC, and injected intravenously into irradiated ia rats. After 48 hr, the recipients' long bones were removed and split longitudinally, and the endosteal surface was scraped. The resulting cellular suspension containing osteoclasts was examined by phase contrast and fluorescence microscopy. Fluorescing mononuclear cells of donor origin that had homed to the bone marrow demonstrated moderate cytoplasmic fluorescence. Approximately 30% of the osteoclasts observed demonstrated light cytoplasmic fluorescence. When cellular pools incapable of curing osteopetrosis (thymocytes) were labeled and injected into ia recipients, no labeled osteoclasts were observed. These studies indicated that pluripotent hemopoietic stem cells, when transplanted into ia hosts, fuse with each other and differentiate into osteoclasts or fuse with pre-existing osteoclasts.

Cellular specificity of the cure for osteopetrosis: Isolation of and treatment with pluripotent hemopoietic stem cells

Osteopetrosis in the ia rat is the result of reduced bone resorption due to abnormal osteoclasts. Studies in the ia mutant have shown that mononuclear cells from normal littermates could cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia rats. Recent studies demonstrated that the Ficoll-Hypaque isolates of spleen, bone marrow, and newborn liver were effective in curing the skeletal disease. These results suggest that the cellular source of the cure is a pluripotent hemopoietic stem cell (PHSC). Goldschneider et al. (1980) demonstrated that stem cells from rat bone marrow relatively large mononuclear cells that are strongly positive for Thy 1.1 antigen. Splenic and bone marrow samples from normal rats were treated with a monoclonal antibody developed against the rat Thy 1.1 antigen and guinea pig complement. This procedure was cytotoxic for 47% of the mononuclear spleen cells and 74% of the mononuclear bone marrow cells. When the Thy 1.1 depleted samples were transplanted into ia littermates, they were ineffective in reversing the osteoclast and bone resorption defects. Using the parameters described by Goldschneider, and fluorescence-activated cell sorting (FACS), the PHSC were isolated from normal rat bone marrow. The isolated cells were assayed for colony forming units — spleen (CFU-S) and were found to contain approximately 65–70% PHSC. Two, 3, and 5-week-old ia rats were given an intraperitoneal injection of normal bone marrow stem cells (1.4 × 10 4 to 7.0 × 10 4 cells). The effects of treatment were evaluated by radiography, by measuring the size of the tibial marrow cavity, and by identifying active (phenotypically normal) osteoclasts at 3 weeks after transplantation. At all of the ages transplanted and at all of the cell numbers tested, the stem cell suspensions were effective in curing the osteopetrotic condition. These positive findings and the fact that the Thy 1.1 depleted cells were ineffective suggests that the PHSC is a cellular source of the cure in the ia mutation.

Stimulation of mucosal mast cell growth in normal and nude rat bone marrow cultures.

Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.

Generation of mucosal mast cells is stimulated in vitro by factors derived from T cells of helminth-infected rats.

The connective tissue of rats, and several other species of mammals, contains two distinct types of mast cells that differ in morphology, histochemical staining properties and location1. One type, frequently called the normal connective tissue mast cell, can be obtained in nearly homogeneous preparation from a mixed cell population in the peritoneal cavity and forms the basis of our knowledge of mast cells. The other type is referred to as the mucosal mast cell because in normal rats it has been observed only in mucosal tissue. Infection with helminth parasites induces an exteNsive accumulation of mast cells and eosinophils in the tissues, and parasites of mucous surfaces, in particular, stimulate a rapid hyperplasia of mucosal mast cells. However, the origin of mucosal mast cells, and their relationship to the connective tissue mast cells is uncertain. We now slow that lymphocytes of helminth-infected rats, on in vitro stimulation with specific antigen, release factors causing pronounced mucosal mastocytosis in normal rat bone marrow cultures.

Regulation of the growth fraction of CFU-S by an inhibitor produced by bone marrow

Humoral factors were extracted from cell suspensions obtained from rat bone marrow and thymus and fractionated using Amicon filters to obtain crude molecular weight bands of 50–100 K and 30–50 K. These fractions were incubated in vitro with haematopoietic stem cells (CFU-S) obtained from drug-treated mice to evaluate their effects on the proliferation of stem cells. An inhibitor was found in the 50–100 K fraction from normal rat bone marrow that would decrease the proportion of stem cells synthesising DNA in the regenerating bone marrow from ∼ 50 to ∼ 10%. This inhibitor was not found in the 30–50 K fraction extracted from rat bone marrow or in the 50–100 K fraction extracted from rat thymus and had no effect on the proportion of committed progenitors (CFU-C) synthesis DNA or the proliferation of mitogen-stimulated lymphocytes. The inhibitor has the same properties as that described by Lord et al. [11].

Junctional structures in haemopoiesis: a study of bone marrow using freeze-fracture and lanthanum impregnation techniques.

Intercellular regions of contact in the haemopoietic compartment of normal rat bone marrow were studied using freeze-fracture and lanthanum tracer techniques. Small adhering junctions (like desmosomes and their variants) were found between haemopoietic and stromal cells but tight, gap or septate junctions could not be identified. These findings are in agreement with the concept that extensive junctional structures may be inconsistent with orderly development of this transient cell system, preventing the delivery of mature cells into the circulation and resulting in ineffective haemopoiesis. Occasionally 'pinching off' of a portion of the cytoplasm of erythroid cells by stromal cells was seen, providing a means for intercellular communication. Structures similar to intercellular bridges responsible for direct intercellular communication were also seen.

Simultaneous flow cytometric DNA and volume measurements of bone marrow cells as sensitive indicator of abnormal proliferation patterns in rat leukemias.

Simultaneous flow cytometric DNA and volume analysis of normal rat bone marrow cells shows three populations of nucleated cells with different mean volume. Each of these populations proliferates in a distinct cell cycle (alpha, beta, gamma). Normally the alpha-cell cycle has the highest amplitude, the beta-cell cycle is intermediate, and the gamma-cell cycle is low. The alpha-cell cycle was very significantly depressed and the beta + gamma-cell cycle was increased in three different rat leukemias (L5222, Shay, BNML), growing on three different rat strains (BDIX, Holtzmann, Brown Norway). The two parameter analysis further revealed that cells of the beta + gamma-cell cycle were slightly hyperdiploid and hypertetraploid in leukemic animals. The decrease of the alpha-cell cycle and the hyperploidies were more sensitive indicators for the abnormal proliferation pattern than the analysis of one parameter DNA distributions which remained within normal limits in all three leukemias.

IgE-B Cell-Generating Factor from Lymph Node Cells of Rats Infected with Nippostrongylus Brasiliensis

Abstract Cell-free culture supernatants (CFS) of mesenteric lymph node cells from Nippostrongylus brasiliensis-infected rats specifically increase the proportion of IgE-bearing cells in normal rat bone marrow cultures without changing the proportion of IgM-bearing cells and IgG2a-bearing cells. Virtually all of the IgE-bearing cells appearing in the culture carried IgM determinants on their surface. Precursors of IgE-bearing cells in normal bone marrow cells were removed by depletion of immunoglobulin-bearing cells. These findings indicate that IgE-B cell-generating factor converts IgM-bearing virgin B cells to IgM-IgE double bearing cells. Pretreatment of normal bone marrow cells with mitomycin C failed to inhibit the generation of IgE-bearing cells by the soluble factor. Indeed, most of IgE-bearing cells generated in cultures of mitomycin C-treated bone marrow cells did not contain 3H-thymidine, which was present during the entire culture period. It appears that DNA synthesis is not required for the differentiation of IgM-bearing cells to IgE-IgM double bearing cells. However, the appearance of IgE-bearing cells in cultures of normal bone marrow cells by CFS was abated by 0.5 µg/ml of actinomycin D or by pretreatment of the cells with pactamyicn. These findings suggest that the observed differentiation involves some process dependent on transcription of DNA and protein synthesis.

Ultrastructural localization of peroxidase in leukocytes of rat bone marrow and blood.

The differentiation of leukocytes in the bone marrow and blood of normal adult male rats was studied by electron microscopy and peroxidase cytochemistry. Tissue samples were fixed in glutaraldehyde, or paraformaldehyde-glutaraldehyde, and incubated in a peroxidase medium containing 3,3'-diaminobenzidine and H2O2 ad pH 7.6. Mature cells of blood were identified, and then the earlier stages of maturation in bone marrow were analyzed. In immature cells of four cell lines, neutrophils, monocytes, basophils, and eosinophils, peroxidase is synthesized and could be demonstrated in the rough endoplasmic reticulum (RER), Golgi complex, and in cytoplasmic granules. Later in maturation, reaction product for peroxidase could not be found in RER or Golgi complex, indicating that peroxidase synthesis had ceased. In two cell lines, neutrophils and monocytes, peroxidase-negative granules were formed, and the mature cells contained two populations of cytochemically distinct granules. All granules of mature eosinophils were peroxidase-positive. In mature basophils, some granules were clearly peroxidase-positive; others displayed variable density, making interpretation uncertain. Mast cells were never seen in blood, but were abundant in bone marrow; peroxidase was never found in their granules by either electron microscopic cytochemistry or a variety of light microscopic methods. Hence, these cells differ from basophils, not only in morphology but also in the enzyme content of their granules.

Assessment of erythrocytic and granulocytic colony formation in an in vivo plasma clot diffusion chamber culture system

Normal rat bone marrow cells seeded into a plasma clot diffusion chamber culture developed into erythrocytic and granulocytic colonies in vivo. Chambers implanted into the peritoneal cavity of normal hosts showed erythrocytic colony numbers reaching an initial peak on day 2, declining on days 3--5, and increasing in a secondary growth phase on day 7. Day 2 colonies were evenly dispersed; day 7 colonies were grouped into discrete areas of bursts. Granulocytic colony numbers reached a peak on day 4 and gradually declined through day 7. Cells in various stages of differentiation could be detected in both colony types. Colony numbers were proportional to the number of marrow cells seeded into the chamber. Host animals treated with phenylhydrazine induced a marked increase in erythrocytic colony numbers and size and a decrease in granulocytic colony formation. Host animals treated with endotoxin suppressed erythrocytic colonies while increasing granulocytic colony size. This method may prove advantageous for the study of hematopoietic colony formation in a physiologic environment.

Assessment of Erythrocytic and Granulocytic Colony Formation in an In Vivo Plasma Clot Diffusion Chamber Culture System

Normal rat bone marrow cells seeded into a plasma clot diffusion chamber culture developed into erythrocytic and granulocytic colonies in vivo. Chambers implanted into the peritoneal cavity of normal hosts showed erythrocytic colony numbers reaching an initial peak on day 2, declining on days 3--5, and increasing in a secondary growth phase on day 7. Day 2 colonies were evenly dispersed; day 7 colonies were grouped into discrete areas of bursts. Granulocytic colony numbers reached a peak on day 4 and gradually declined through day 7. Cells in various stages of differentiation could be detected in both colony types. Colony numbers were proportional to the number of marrow cells seeded into the chamber. Host animals treated with phenylhydrazine induced a marked increase in erythrocytic colony numbers and size and a decrease in granulocytic colony formation. Host animals treated with endotoxin suppressed erythrocytic colonies while increasing granulocytic colony size. This method may prove advantageous for the study of hematopoietic colony formation in a physiologic environment.

In Vitro Erythroid Colony Formation in Acute Myelogenous Leukemia in the Rat 2

Erythroid colonies were grown in vitro in plasma clot cultures. Normal adult rat bone marrow responded to exogenous erythropoietin with the formation of an average of 2 colonies/10(3) cells plated. No erythroid colonies were observed in cultured normal spleen preparations. Shay chloro-leukemia cells administered iv induced an acute myelogenous leukemia. During the progressive stages of the disease, the numbers of erythrocyte colony forming units (CFU-E) in the marrow decreased; concomitantly, these progenitors appeared in leukemic spleen cultures. Paralleling changes in CFU-E, the numbers of nucleated red blood cells in the marrow declined but increased in the leukemic spleen. However, compensatory spleen erythropoiesis was transient, due to continued leukemia cell colonization. The loss of erythroid progenitor cells from the bone marrow played a significant role in the anemia associated with this leukemia.

The anti-folate effect of methionine on bone marrow of normal and vitamin B12 deficient rats.

The effects of dietary vitamin B12 and methionine deficiency, and the in vitro addition of methionine, homocysteine, or folic acid on the methylation of dUMP to dTMP were studied in rat bone marrow culture. Vitamin B12 or methionine deficiency had no effect on the methylation reaction or on bone marrow folate levels although the vitamin B12 content in bone marrow was reduced in vitamin B12 deficiency. In vitro addition of vitamin B12 or folic acid also had no effect on the methylation of dUMP. In vitro addition of methionine reduced the methylation of dUMP and increased the proportion of 5-methyltetrahydrofolate at the expense of other folate coenzymes. The reason for this 'anti-folate' effect of methionine, which is the opposite to that found in liver, was not clear. The presence of 5,10-methylenetetrahydrofolate reductase and 5-methyltetrahydrofolate-homocysteine methyltransferase were confirmed in rat bone marrow and they were inhibited by S-adenosylmethionine and methionine, respectively, in a similar fashion to that found with the liver enzymes. Homocysteine had no effect on the proportions of the various folate coenzymes in bone marrow but did inhibit the incorporation of deoxyuridine and deoxythymidine into DNA. It appeared that homocysteine exerted at a non-folate dependent step beyond the formation of dTMP.

Ultrastructural Study of Bone Marrow‐Derived Granulocytic Colonies in Diffusion Chambers in Rats

An ultrastructural study of diffusion chamber granulocytic colonies is reported. The colonies were derived from normal rat bone marrow cells and the chambers were inserted into the peritoneal cavity of lethally irradiated rats. The colonies were processed en bloc for electron microscopy enabling observation of cellular relationship to be made. Ultrastructural analysis of colonies disclosed normal maturing cells at all stages of maturation, with the exception of occasional granulocytes on neutrophil type that presented narrow strips of chromatin ('nuclear blebs'). The cells in colonies generally occurred in close apposition to one another and to occasional macrophages. This organization possible enhanced transmission of factors of maturation. Advantages of diffusion chamber haematopoietic culture are discussed; among the most important are the occurrence of fibroblasts originating from implanted marrow cells and the local formation of fibrin within the chambers.

Suppression and regeneration of rat bone marrow under the influence of methotrexate. An EM study.

Adult male rats were given daily injections of methotrexate for 12 days, during which time specimens of femoral bone marrow were taken for correlative light- and electron-microscopic study. After 12 days methotrexate was discontinued and specimens were taken the marrow recovered from the effect of methotrexate. Normal rat bone marrow was used as a basis of comparison. As methotrexate was administered, cell division ceased and the immature erythroblasts and myelocytes proceeded in the maturational process and a rapid hypocellularity of the marrow developed. Eosinophilic leukocytes, lymphocytes, large fat cells, erythrocytes, and phagocytic reticuloendothelial cells were the only formed elements remaining after 12 days of methotrexate treatment. Two days after methotrexate was discontinued, myeloblasts, myelocytes and proerythroblasts were plentiful, and on the third day the marrow exhibited an erythroid hyperplasia. By the eighth day all cell types were present though the marrow still was hypocellular. 14 days post methotrexate the marrow returned to normal.

Eosinophils in the bone marrow of normal and cortisol-treated rats. Quantitative and autoradiographic studies.

Absolute counts of the eosinophil cells of the blood and bone marrow were carried out in a haemocytometer and combined with autoradiographic studies of smears of blood and bone‐marrow suspensions from normal rats and cortisol‐treated rats. A certain parallelism was found between the number of circulating eosinophil leucocytes and the eosinophils of the bone marrow. Calculations showed that the bone marrow contains more than 50 times as many eosinophils as the blood. Single injections of Cortisol (5 mg intraperitoneally) as well as repeated injections of Cortisol (5 mg every 6 hours for up to 3 days) induced blood and bone‐marrow eosinopenia. The decrease in the number of eosinophils in the bone marrow appears to be due primarily to a decrease of mature (non‐prolifera ting) eosinophils. During repeated injections there was continued proliferation of eosinophil cells in the bone marrow, but mitotic activity was reduced. Appearances like those in the bone marrow have previously been observed in the spleen (Bro‐Rasmussen 1972 and 1973).

MECHANISM OF EOSINOPHILIA

ABSTRACTEosinophil leucocyte production was studied in the bone marrow of normal rats and rats given single injections of Trichinella spiralis larvae which stimulated eosinopoiesis. the development sequence of eosinophils in the bone marrow was based on morphological criteria combined with studies of the extent of eosinophil labelling after injections of tritiated thymidine. the proliferative compartment contained at least three recognizable steps in eosinophil development which were defined.There was a delay of 23 hr after injection of larvae before the proportion of eosinophils in the bone marrow increased and it had doubled by 49 hr. the mitotic index increased by a factor of 3 after stimulation. Estimates of the cell cycle parameters were made for marrow eosinophils 1‐3 days after stimulation, using the technique of analysing labelled mitoses. the results were compared with a similar group of normal rats, and were processed by using a computer program. Marrow eosinophil cell cycle time was 30 hr in normal rats and 9 hr in stimulated rats, and this acceleration was associated with a reduced spread of cell cycle times. the number of eosinophil cell divisions and the transit times for each compartment in normal and stimulated rats were estimated. This showed that the stimulus may have resulted in five or six additional divisions among the youngest eosinophils in the dividing compartment. From these figures an outline of eosinopoiesis in the marrow of normal and stimulated rats is proposed.

Control of Granulopoiesis in Bone Marrow of Normal Albino Rats by a Maturation Factor Isolated from Rat Serum

Control of Granulopoiesis in Bone Marrow of Normal Albino Rats by a Maturation Factor Isolated from Rat Serum

The response of RNA synthesis to mitotic regulation in the erythron.

AbstractMitotic homeostasis in the erythron appears to be controlled by a humoral feedback mechanism consisting of a tissue‐specific mitotic inhibitor, the erythrocytic chalone, and a tissue‐specific mitotic stimulator. The present report is an account of the way in which these two regulatory factors affect RNA synthesis in normal rat bone marrow cells in vitro. The responses were measured in terms of 3H‐uridine incorporation into the target cells. The primary action of the erythrocytic chalone on 3H‐uridine incorporation was manifested as a rapid and strong inhibition. The initial inhibition was reversible and was followed, after a few hours' incubation, by an increased rate of 3H‐uridine incorporation, which ultimately led to complete compensation of the inhibition. The tissue specificity of these findings was demonstrated indirectly by means of polycythemic target bone marrow, on which the erythrocytic chalone had only a negligible effect. The effect of the mitotic stimulator on 3H‐uridine incorporation in the cells was invariably a stimulation, which showed a slight tendency to increase with incubation time. The results demonstrate that the effects of the mitotic regulatory factors are not restricted to DNA metabolism alone but can also be elicited within other metabolic processes in the cell.