Functional neutrophils from long-term murine bone marrow cell cultures

Murine bone marrow cell cultures that had been established for up to 26 weeks were harvested each week and found to provide functional neutrophils. Leukocytes harvested from the cultures were enriched for neutrophils using discontinuous Percoll density gradients. These cells mounted a chemiluminescence response to Proteus mirabilis in the presence of normal mouse serum (NMS). They killed several NMS-opsonised bacterial species, an activity that was blocked by a monoclonal antibody to the C3 receptor of mouse neutrophils. Cultured bone marrow neutrophils expressed both Fc and C3 receptors. C3 receptor expression could be augmented by exposure to the chemotactic peptide f-Met-Leu-Phe. We conclude that murine bone marrow cell cultures provide a useful source of functional neutrophils, and that their productivity can be sustained in long-term culture. As their receptor expression can be augmented from the resting state by exogenous stimuli, they represent a useful cell source in studies of neutrophil activation.     

Establishment of long-term CD154-dependent porcine B-cell cultures

Cells of the B-cell lineage play an essential part in the immune response, not only as the producers of antigen-specific antibodies, but also as antigen-presenting cells. Unlike T cells, however, the establishment of long-term normal B-cell lines has proved to be exceedingly difficult. In this paper we demonstrate that cell membrane-expressed CD154 (CD40 ligand) is able to support the continual growth of porcine mesenteric lymph node B-cell cultures for more than 4 months without the addition of exogenous cytokines, such as interleukin-4 (IL-4). Addition of IL-4, but not interferon-gamma (IFN-gamma) or IL-13, to these cultures enhanced proliferation, as, to a lesser extent, did addition of IL-2. Interestingly, however, whilst IFN-gamma-supplemented cultures largely consisted of immunoglobulin M (IgM)-positive cells, cultures with IL-13 or IL-4 contained a significantly increased proportion of IgG-positive cells.     

An investigation into the B lymphopoietic capacity of long-term bone marrow cultures

A suspension of nucleated femoral (BALB/c x CBA) bone marrow cells was inoculated, either into glass flasks or into flasks coated with a preformed syngeneic marrow derived monolayer, and cultured for long periods in media supplemented with hydrocortisone sodium succinate. Long term analysis of the lymphoid status of these cultures was then carried out whilst they were producing CFU-S cells. The results show that T cells expressing the Thy 1 marker were present for up to 10 weeks, surface immunoglobulin positive (sIgM+) B cells were present for at least 6 weeks, whereas pre-B cells were present for no longer than one week. Pre-B, B and T cells were all present at levels well below those found in normal healthy bone marrow. When cultures were free of both B and pre-B lymphocytes, the cells collected from them were used to reconstitute lethally irradiated mice. Reconstitution was of the type (A x B) leads to A and approximately 2 months elapsed before reconstituted animals were analysed. The results show that despite the loss of pre-B cells from these cultures, precursors of B lymphocytes at earlier stages of differentiation were present for long periods, but were only capable of differentiation into sIgM+ B cells in vivo.     

Suppression of polyclonal B cell proliferation mediated by supernatants from human myeloma bone marrow cell cultures.

For functional characterization and semi-quantitative estimation of soluble regulator factors influencing polyclonal B cell proliferation and differentiation, we established two assays. One of the assays measures enhancement or inhibition of proliferation from purified human spleen B lymphocytes, and the other one the effect of soluble factors on CESS-cell differentiation. We found no difference concerning regulator factors for B cell differentiation between bone marrow cell culture supernatants from multiple myeloma (MM) patients and from controls, whereas significantly higher suppressor activity on polyclonal B cell proliferation could be detected in the former group of supernatants. The extent of such determined suppressor activity in vitro correlated with the amount of polyclonal serum IgM of the corresponding patients. These results indicate that one or several soluble suppressor factors may be involved in immunoregulatory mechanisms responsible for the humoral immunodeficiency observed in MM patients.     

Bone marrow hematopoiesis in polymer composite implants in long-term bone marrow cultures

The morphological composition of cells on implants made of different composition materials was studied on the model of long-term bone marrow cultures at cultivation for 28 days. Three samples of materials were investigated: polymethyl methacrylate (PMMC), PMMC with hydroxyapatite (HA) and PMMC with HA and subsequent processing with supercritical carbon dioxide. A gradual rise in total number of cells was observed on the implants for 4 weeks as well as increasing number of stromal and hemopoietic cells. With increasing time of cultivation the share of stromal cells was growing while the percent of hemopoietic cells fell. The greater number of hemopoietic cells was observed on the samples of the implants made from composites containing HA vs implants made from the initial PMMC.     

Stromal components in rat bone marrow frozen sections compared to long-term rat bone marrow cultures

The haematopoietic microenvironment is believed to play an important role in controlling the haematopoietic process. Ultrastructural studies have shown that the haematopoietic stroma is composed of cellular as well as extracellular components. Relatively little is known about the distribution of the different stromal components in the bone marrow. The knowledge on the bone marrow microenvironment is mainly based on studies in which in vitro long-term bone marrow cultures have been used. Although this culture system offers a unique possibility to study haematopoietic in vitro, it does not fully represent the complexity of intact bone marrow.In the present study we describe the immunohistochemical distribution of different cellular and extracellular stromal components in frozen sections of rat bone marrow as well as in long-term bone marrow cultures, in order to compare the haematopoietic microenvironment used in in vitro studies in the in situ situation. We found that in situ a specific compartmentalization of stromal components exists in the bone marrow. Under culture conditions however, most stromal components are indeed present but the architecture present in the in situ situation had almost completely disappeared. The interaction between the different stromal elements was studied in the long-term bone marrow cultures. It appeared that under the chosen conditions, nodules were formed with a core of reticular cells and extracellular matrix. In close contact with this core immature macrophages appeared to proliferate and differentiate into mature, non-dividing macrophages.     

Early B cell precursors in long-term bone marrow culture: selective development in the bone marrow of irradiated recipients

The primary site for the growth and differentiation of B cell precursors in irradiated recipient mice was investigated. Bone marrow (BM) cells from lipopolysaccharide (LPS)-responder C57BL/6 mice were transferred into irradiated LPS-nonresponder C57BL/10ScCr mice, and the generation of donor-derived B cells in the recipient was monitored by determining the immunoglobulin-producing cells developed in the LPS-stimulated cultures of recipient's spleen cells as well as BM cells. As previously stated, the transfer of fresh BM cells resulted in the development of LPS-reactive cells both in spleen and BM simultaneously. On the other hand, when long-term cultured BM cells which were shown to be devoid of B cells and pre-B cells were used as the donor cells, the development of LPS-reactive cells was first observed only in BM, and subsequently in both BM and spleen. The failure to detect LPS-reactive cells in the spleen in the early phase, or day 11 after irradiation and reconstitution, was not attributed to the culture condition or the existence of suppressive activity in day 11 spleen cells. These results indicate that B cell precursors lodge only in the BM of irradiated recipients, grow and differentiate in the same place, and then the differentiated progeny migrate to peripheral lymphoid organs.     

稳定转染增强型绿色荧光蛋白大鼠骨髓间充质干细胞系的建立

背景：利用间充质干细胞或含有治疗因子的干细胞进行有选择性的杀伤肿瘤细胞是一种有前途的治疗方法。 目的：建立含稳定转染增强型绿色荧光蛋白的大鼠骨髓间充质干细胞系。 方法：通过脂质体介导慢病毒质粒pVector-EGFP、pHelper、Envelope 共转染293T细胞完成载体病毒构建,以实时荧光定量PCR检测慢病毒滴度;取对数生长期的SD大鼠骨髓间充质干细胞,以复感染指数MOI值0,5,10,15,20加入携带报告基因增强型绿色荧光蛋白的慢病毒载体稀释液,72 h后观察各组增强型绿色荧光蛋白的表达效率及阳性转染率。 结果与结论：携带增强型绿色荧光蛋白的慢病毒载体系统转染293T细胞能够正确表达,滴度为1×10⁸ TU/mL。包装好的病毒颗粒转染大鼠骨髓间充质干细胞二三天后,各孔均有增强型绿色荧光蛋白的表达。MOI值从0增至10,细胞的阳性表达率逐渐提高(P < 0.05),MOI值为10的组能获得>70%的转染率,但MOI值从10增至20,转染率变化不明显。说明以MOI值为10的滴度将慢病毒载体可将外源基因高效转入大鼠骨髓间充质干细胞内,建立含稳定转染增强型绿色荧光蛋白的大鼠骨髓间充质干细胞系。     

Recent studies of the hematopoietic microenvironment in long-term bone marrow cultures

Supported by research grants from the National Institutes of Health — CA39851. CA40818 and PO1HD19767.     

Delayed and deficient establishment of the long-term bone marrow plasma cell pool during early life

Early life antibody responses are characterized by a rapid decline, such that antigen-specific IgG antibodies decline to baseline levels within months following infant immunization. This generic observation remains unexplained. Here, we have analyzed the induction and organ-localization of antigen-specific IgG antibody-secreting cells (ASC) following immunization of 1-week-old or adult BALB/c mice with tetanus toxoid (TT), a T-dependent antigen. Early life priming induced only slightly lower numbers of TT-specific IgG ASC in the spleen, and these reached adult levels following repeat immunization. In contrast, early life immunization generated much fewer bone marrow plasma cells than in adults, even after boosting. A similar limitation of the natural development of the bone marrow pool of ASC was observed. Transfer experiments with adult or early life spleen ASC indicated limited homing of TT-specific adult ASC to the bone marrow of 4-week-old mice as compared to adult recipients, whereas homing patterns were similar when early life or adult ASC were transferred into adult recipients. These observations suggest that a limited bone marrow B cell homing capacity and, as a result, relatively deficient bone marrow ASC responses, are critical factors which may explain the limited persistence of IgG antibodies to T-dependent antigens in early life.     

Effect of opioid peptide methionine-enkephalin in long-term cultures of human bone marrow

Methionine enkephalin, an opioid peptide belonging to the family of neuropeptides, has been shown to function as a neurotransmitter, hormone and growth factor. The present work explored its effects in long-term culture of bone marrow cells, harvested from a patient with acute lymphoid leukemia (ALL-L3) in the second complete remission. Nine cultivation flasks were established and maintained for five weeks, with medium renewal once a week. At each re-feeding, methionine-enkephalin was added to the cultures in final concentrations 10(-8), 10(-10) or 10(-12) M, and granulocyte-macrophage progenitor cells (GM-CFU) were determined among the harvested, nonadherent cell populations. The total number of nonadherent cells was 8% to 42% higher in the treated cultures than in the control, nontreated cultures, and the GM-CFU counts were three to four times higher. Those changes, although evident, did not reach statistical significance because of the small group sizes. In 1 of 9 cultures the adherent cell layer was atypical, the cell population consisted of small cells resembling the lymphoblasts, and the cell count was 2-8 times higher than in the controls. That aberrant culture has presumably arisen from residual leukemic cells remaining in the bone marrow after chemotherapy. The findings support the idea that opioid peptides, including methionine-enkephalin, participate in regulation of hematopoiesis. Two mechanisms may have accounted for the observed effects of enkephalin on cultured bone marrow cells: an indirect action, via interleukins secreted from the stromal cells upon stimulation of the opioid receptors, or a direct action on hematopoietic precursors.