Concentration of mast-cell progenitors in bone marrow,spleen, and blood of mice determined by limiting dilution analysis

When hematopoietic cells of congenic +/+ mice were injected into the skin of genetically mast-cell-depleted (WB × C57BL/6)F₁-W/W^v mice, mast cells appeared at the injection site. The donor origin of developing mast cells was confirmed by using granules of C57BL/6-bgl/bgl mice as a marker. When the number of injected cells was decreased, the proportion of injection sites at which mast cells did not appear increased according to the expected frequency of null response in a Poisson distribution. Therefore, such proportions were used to calculate the concentration of mast-cell precursors in the bone marrow, spleen, and peripheral blood. The relative concentration of mast-cell precursors in these tissues was similar to that of spleen-colony-forming cells. The present method seems useful as a semiquantitative in vivo assay for a population of progenitor cells which are committed to differentiate into mast cells.     

Limiting dilution analysis of the stem cells for T cell lineage

Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.     

Hemopoiesis in spleen and bone marrow cultures

Four endothelial cell clones derived from adult bovine aorta were examined with respect to their proliferative characteristics in vitro. Three of these clones, derived in the absence of fibroblast growth factor (FGF), displayed variable basal proliferative rates. One of these non-FGF derived clones grew at a maximal rate which could not be further enhanced with FGF. The other two clones grew at a suboptimal rate which was stimulated by low doses of FGF (10-50 ng/ml) and inhibited by higher doses (100-250 ng/ml). The fourth clone, derived in the presence of FGF, was stimulated by FGF in a dose-dependent manner (10-250 ng/ml) and was not growth inhibited at high FGF concentrations (250-1,000 ng/ml). Growth of all four clones on extracellular matrix (ECM) derived from bovine aortic smooth muscle (BASM) cells was optimal in the absence of FGF. ECM-coated dishes also significantly increased the sensitivity of all clones by at least fivefold to mitogenic stimulation by serum. The proliferative lifespans of the clones ranged between 60 and 120 generations with the most actively proliferating clones attaining the greatest lifespan. Continuous subculture of two of the endothelial clones in the presence of FGF or on ECM-coated dishes did not induce a dependence of the cells on either factor for subsequent growth in its absence. The results indicate that aortic endothelial cells display considerable clonal variability in ther basal proliferative rate and in their response to FGF. This clonal variability is not observed when the cells are maintained on ECM-coated dishes derived from vascular smooth muscle cells.     

Cell surface characteristics and DNA content of macrophages in murine bone marrow cultures

Summary An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied immediately thereafter with the SEM part of the instrument. The cells were divided into six groups according to the number of microvilli and/or microridges present on their surface. A proportion of macrophages showed a DNA content more than occurs in diploid cells, which could indicate a future division. No special surface morphology could be detected in this cell type.     

Generation of mesenchymal stem cell lines from murine bone marrow

Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages.     

Renewal of hematopoietic stem cell clones in long-term bone marrow cultures

In long-term cultures of murine bone marrow, clonal succession of hemopoietic cells was observed as measured by karyologic analysis. There were high oscillations in self-renewal of CFUs in the cultures. A close correlation between the CFUmix karyotype and mitotic non-adherent cells in culture (but not between these cell types and CFUs) was revealed.     

Diffusion chamber and spleen colony assay of murine haematopoietic stem cells

Mouse bone marrow cells have been cultured in diffusion chambers and their capacity to form spleen colonies in irradiated mice investigated after different culture periods. The number of spleen colony-forming units (CFU) in the chambers decreased during the first day of culture. The number then increased rapidly to a level significantly above the original chamber value on the third to fifth day of culture. By that time large numbers of granulocytes and macrophages had also appeared. Histological examination of spleen colonies showed that prior culturing did not alter the ratio between the different types of colonies. Cultured bone marrow cells which were transferred to new chambers retained granulopoietic capacity. This capacity increased between the first and second day of primary culturing. At this time hydroxyurea injections to chamber hosts revealed that the progenitor cells were proliferating. The results show that the granulopoietic progenitor cells of the chambers are stem cells, and that one progenitor cell type is identical with the CFU.     

Pluripotential stem cell replication in continuous human, prosimian, and murine bone marrow culture

Prolonged replication of pluripotential stem cells and committed progenitor cells is sustained for prolonged periods in a murine marrow culture system. Alterations in stem cell replication and differentiation are observed after infection of the cultures with Friend virus and Kirsten sarcoma virus consistent with transformation of pluripotential stem cells in the first case and transformation of the macrophage component of the hemopoietic microenvironment in the second. Prolonged myelopoiesis and CFU-c proliferation was also observed in continuous human and prosimian marrow cultures, suggesting the applicability of this technique for analysis of stem cell control and in vitro leukemogenesis in species other than the mouse.     

Generation of natural killer cell lines from murine long-term bone marrow cultures

Functionally active natural killer (NK) cells with the ability to lyse 51Cr-labeled YAC-1 lymphoma target cells are no longer detectable by 1 wk of culture in cultured marrow cells harvested from Dexter-type long-term marrow cultures (LMC). Interferon, which enhances NK cell-mediated target cell lysis, fails to induce NK activity from LMC cells even at high effector to target cell ratios. However, such LMC cells, when placed in secondary cultures in the presence of Con A-splenic leukocyte-conditioned medium (spleen-CM) generated a population of cells with NK activity within 1 wk. Kinetic studies showed that the generation of NK activity was not due simply to proliferation of a few surviving NK cells, but suggested derivation from NK precursors through clonal expansion and functional maturation. This NK activity was further shown to be associated with a subpopulation of cells bearing surface Thy-1, Ly-5, and NK-1 as well as asialo-GM1 antigens but lacking Ly-1 antigen. The expression of Ly-2 antigen, however, was variable. Electron microscopy studies of isolated asialo-GM1-positive cells showed a uniform lymphoblastoid morphology with large cytoplasmic to nuclear ratios and prominent electron dense cytoplasmic granules characteristic of large granular lymphocytes. In support of the NK nature of such cultured cells was the ability of anti-asialo-GM1 and complement to abrogate, and of interferon to augment, target cell lysis. Isolated cell lines also showed target selectivity similar to NK cells. The implications of the studies on further analysis of the nature of NK precursors is discussed.     

Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture

Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C57B1 mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.     

Limiting dilution clonal assay of human bone marrow hematopoietic progenitors (granulocytes-macrophages)

We have developed a limiting dilution assay for human bone marrow hematopoietic precursor (granulocytes-macrophages) in microwells. Bone marrow cells were fractionated by discontinuous Percoll gradients and diluted in culture medium containing colony-stimulating factors. They were diluted and cultured in medium containing colony-stimulating factor and 10(-5) M hydrocortisone in microwells over a range of cell densities that allowed calculation of the frequency of growth-inducible precursors. After 10 days, the wells were examined for clonal growth. Clonal proliferation followed the single-hit model of the Poisson distribution. More progenitors were detected in this assay than in simultaneous methylcellulose colony assays or agar cultures. Thymidine suicide experiments led to an increase in the frequency of progenitors detected in this assay, but a decrease in the frequency of methylcellulose colonies. This system may detect additional, less mature progenitors than are detected in semisolid culture systems.     

Stimulating factors and cell recruitment in murine bone marrow stem cells and EMT6 tumours.

The role of a stimulating factor in cell recruitment and the kinetics of its secretion were investigated by in vivo and in vitro techniques. The association of these two methods made it possible to demonstrate that a non-cycling population liberates a factor which in turn stimulates quiescent bone marrow stem cells into DNA synthesis. Moreover, it seems that undamaged cells are capable of secreting this factor. A stimulating factor responsible for cell recruitment was also demonstrated in an experimental EMT6 tumour and the kinetics of its secretion reported.     

Generation of osteoclasts in cultures of rabbit bone marrow and spleen cells

The primary and specific function of the osteoclast is the resorption of bone. We have applied this criterion, and a monoclonal antibody that binds specifically to osteoclasts, to cultures of tissues that may contain osteoclastic precursors. Bone marrow and spleen cells were incubated for up to 4 weeks in the presence or absence of parathyroid hormone, interleukin 1, or 1,25(OH)2 vitamin D3, on plastic coverslips or slices of devitalised bone. Osteoclasts (as judged by the presence of resorption cavities and the appearance of monoclonal antibody-positive cells) did not develop in cultures incubated without added hormones, nor in cultures containing parathyroid hormone or interleukin 1, but were regularly observed when bone marrow cells were incubated with 1,25(OH)2 vitamin D3. Although multinucleate giant cells were common after incubation, especially in the presence 1,25(OH)2 vitamin D3, monoclonal antibody bound not to these cells but to a minor and distinctive population of mononuclear cells and cells of low multinuclearity. We found no excavations and no monoclonal antibody-positive cells after incubation of peritoneal macrophages with 1,25(OH)2D3. These results provide direct evidence of osteoclastic function arising in cultures of haemopoietic tissues.     

A self-renewing, bipotential erythroid/mast cell progenitor in continuous cultures of normal murine bone marrow

We have established permanent lines of nonadherent cells from fresh normal mouse bone marrow in media containing pokeweed mitogen-stimulated spleen cell conditioned medium (PWSCM). These lines continuously produced erythropoietic progenitor cells (detected by their ability to form erythroid bursts in semi-solid medium containing erythropoietin) together with cells having characteristics of the mast cell lineage (as demonstrated by metachromatic staining with toluidine blue, histamine content and membrane receptors for IgE). Sixteen such cell lines have been established in sixteen attempts. Cloning experiments were carried out to determine the nature of the progenitor cell(s) responsible for the permanence of these cultures. When cells were cultured in methylcellulose medium containing PWSCM, colonies were observed which reached macroscopic size after 4 weeks of incubation. Replating of individual primary colonies resulted in secondary colony formation, indicating the presence of progenitor cells with self-renewal potential. Forty-seven primary colonies were picked and their cells were suspended in liquid culture medium containing PWSCM. Of these, twenty-one could be expanded to establish permanently growing sublines. Sixteen of these sublines were found to be composed of both erythroid progenitors and mast cells. In five sublines only mast cells could be seen; none of the sublines appeared to be purely erythroid. Karyotypic analysis of mast cells and of erythroid cells of seven sublines derived from individual colonies which arose in cocultures of male and female cells revealed that the mast cells and erythroid cells were both of the same sex in each of the seven sublines; this demonstrates the single cell origin of each colony and of the two lineages derived from it. We conclude that these nonadherent, factor-dependent cell lines are maintained by self-renewal and differentiation of bipotential progenitor cells apparently restricted to the erythroid and mast cell lineages.     

骨髓基质干细胞的分离纯化及培养

目的 建立骨髓基质干细胞(MSCs)良好的分离纯化和培养方法。方法 将小鼠骨髓基质干细胞自殷骨中分离,应用贴壁选择法结合细胞克隆挑选法进行分离纯化,应用细胞生长因子(EGF和PDGF-BB)刺激法进行MSCs的体外培养和传代,倒置显微镜下观察分离培养的细胞并照像记录。结果,培养获得了纯化的呈梭形成纤雏样细胞的骨髓基质干细胞。在生长因子EGF和PDGF-BB的共同作用下,传代MSCs生长旺盛,形态均一。结论 该方法是简便高效的骨髓基质干细胞的分离纯化和培养方法。     

Effects of cyclophosphamide on murine bone marrow and splenic megakaryocyte-CFC,granulocyte-macrophage-CFC,and peripheral blood cell levels

The effect of cyclophosphamide (CY) on megakaryocytopoiesis in mice was examined with assays of megakaryocyte colony-forming cells (Meg-CFC) in bone marrow and spleen and simultaneous determinations of peripheral blood counts, after a single intraperitoneal dose (200 mg/kg) of CY. Significant rebound thrombocytosis (170% of normal) occurred at day 11 after injection with CY, although only modest preceding thrombocytopenia (70% of normal) was observed. After an initial 3–5-day period of suppression, total megakaryocyte colony-forming cells (Meg-CFC) in both bone marrow and spleen of CY-treated mice demonstrated rebound increases at 5 and 7 days, respectively, after administration of the drug. Granulocyte-macrophage colony-forming cells (GM-CFC) exhibited alterations which were similar to those of Meg-CFC, suggesting similar sensitivities of Meg-CFC and GM-CFC to CY. The increase in Meg-CFC in both bone marrow and spleen preceded development of thrombocytosis by 4–6 days. This suggests that increased platelet counts in CY-treated mice are attributable, at least in part, to alterations in feedback mechanisms which control megakaryocytopoiesis, with resultant stimulation of the megakaryocyte progenitor compartment.