Growth of human acute myeloblastic leukemic (aml) cells in vitro.

Human normal and granulocytic leukemic progenitor cells produce colonies and clusters when cultured in the plasma clot system in vitro. The number of colonies formed and their cellular composition was comparable to that reported for the system (soft agar) usually used for these studies. The plasma clot system has the advantage of permitting in situ morphologic and cytochemical characterizaion of the cells within the colonies. A comparison was also made of the growth of leukemic cells in suspension cultures placed within conventional and modified Marbrook flasks. These studies demonstrated no advantage for the Marbrook system in cultures incubated for up to 7 days.     

TPEN selectively eliminates lymphoblastic B cells from bone marrow pediatric acute lymphoblastic leukemia patients

BioMetals - B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by the abnormal proliferation and accumulation of immature B-lymphoblasts arrested at various stages...     

Immunological and functional characteristics of the blast cells in acute monoblastic and acute myeloblastic leukemias

The increase in the expression of membrane receptors of monoblast cells to the Fc-fragment of immunoglobulins of classes IgG, IgA, IgM, to the C3-component of complement and FcH receptor in 8 patients with acute monoblastic leukemia and in 3 patients with acute myelomonoblastic leukemia, compared to results obtained for 11 patients with acute myeloblastic leukemia. Leukemic cells in the cases of acute myelomonoblastic and monoblastic leukemia maintained such properties of the phagocytosis (restoring Nitro-blue tetrazolium). Distinctions in degrees of expression of membrane receptors and functional activity of monoblastic and myeloblastic cells may be used as criteria of differential diagnosis of acute monoblastic and acute myeloblastic leukemia.     

Natural killer cells generated from bone marrow culture

By using anti-Nk-1 antiserum, we detected a significant proportion of Nk-1+ cells in bone marrow (BM) with low lytic activity that can be slightly enhanced with interferon (IF). These BM Nk-1+ cells also bind to YAC-1 cells. Because Nk-1 antigen has been found to mark NK cells, BM appears to harbor immature precursors to natural killer (NK) cells. We therefore used concanavalin A (Con A)-conditioned medium to culture BM cells to induce differentiation of the putative NK precursors. After 3 to 4 days in culture, cytotoxic activity to YAC, that peaked at 6 to 7 days, was consistently generated, and the activity still could be detected after 10 to 14 days in culture. In contrast, experiments using spleen cultures, performed in a similar manner, showed peak activity after 4 to 5 days and the activity declined thereafter. The cytotoxic activity of cultured BM cells was also higher than that of cultured spleen cells. Cultures of BM cells from old mice have good cytotoxic activity. The cytotoxic cells generated were Nk-1+ and Qa-5+. Furthermore, these culture conditions did not maintain the proliferation of CFU-C cells.     

The effect of vitamin D3 metabolites on normal and leukemic bone marrow cells in vitro

We studied the effects of 1,25-dihydroxyvitamin D3 and other metabolites of vitamin D3 on the maturation in liquid culture and on colony formation in semisolid media of marrow and buffy coat cells from patients with myeloid leukemias and from normal individuals. After incubation with 1,25-dihydroxy-vitamin D3, a proportion of both normal and leukemic myeloid cells resembled cells of the monocyte-macrophage lineage; these cells expressed alpha-naphthylacetate esterase and were able to phagocytize and kill candida organisms. When granulocyte-macrophage progenitor cells (CFU-GM) were incubated with 1,25-dihydroxyvitamin D3, the number of monocyte-macrophage colonies was increased and the number of granulocyte colonies was reduced; megakaryocyte colony formation (CFU-Mk) was inhibited substantially; and there was no effect on erythroid (BFU-E) or multilineage (CFU-GEMM) progenitor cell colony formation. We propose that 1,25-dihydroxyvitamin D3 may induce cells that are normally committed to differentiate along the granulocytic pathways to differentiate instead along the monocyte-macrophage pathway. If these in vitro observations reflect the in vivo activity of 1,25-dihydroxyvitamin D3, it may be involved in the modulation of collagen deposits in the bone marrow.     

Control of normal differentiation of myeloid leukemic cells. XII. Isolation of normal myeloid colony-forming cells from bone marrow and the sequence of differentiation to mature granulocytes in normal and D+ myeloid leukemic cells

An enriched population of early myeloid cells has been obtained from normal mouse bone marrow by injection of mice with sodium caseinate and the removal of cells with C3 (EAC) rosettes by Ficoll-Hypaque density centrifugation. This enriched population had no EAC or Fc (EA) rosettes and contained 87% early myeloid cells stained for myeloperoxidase and/or AS-D-chloroacetate esterase, 7% cells in later stages (ring forms) of myeloid differentiation and 6% unstained cells, 2% of which were small lymphocytes. After seeding in agar with the macrophage and granulocyte inducer MGI, the enriched population showed a cloning efficiency of 14% when removed from the animal and of 24% after one day in mass culture. Both the enriched and the unfractionated bone marrow cells gave the same proportion of macrophage and granulocyte colonies. The normal early myeloid cells were induced to differentiate by MGI in mass culture in liquid medium to mature granulocytes and macrophages. The sequence of granulocyte differentiation was the formation of EA and EAC rosettes followed by the synthesis and secretion of lysozyme and morphological differentiation to mature cells. D⁺ myeloid leukemic cells with no EA or EAC rosettes had a similar morphology to normal early myeloid cells and showed the same sequence of differentiation. The induction of EA and EAC rosettes occurred at the same time in both the normal and D⁺ leukemic cells, but lysozyme synthesis and the formation of mature granulocytes was induced later in the leukemic than in the normal cells. The results indicate that selection for non-rosette-forming normal early myeloid cells also selected for myeloid colony forming cells, that these normal early myeloid cells can form colonies with differentiation to macrophages and granulocytes, that normal and D⁺ myeloid leukemic cells have a similar sequence of differentiation and that the normal cells had a greater sensitivity for the formation of mature cells by MGI.     

Characterization of cytotoxic cells generated from bone marrow culture

Bone marrow (BM) harbors precursors (Pre-NK) to NK cells. Recently, we devised an in vitro culture system that induces differentiation of the presumptive BM Pre-NK cells into cytotoxic cells to YAC in the presence of rat concanavalin A (Con A) conditioned medium. We have now analyzed the antigenic phenotype of the effector cells, precursor cells, and the target specificity of these cytotoxic cells. The cytotoxic cells had antigenic profiles similar to endogenous NK cells with the exception of Lyt-2 antigen. They are strongly positive for Qa-5, Thy-1, and partially positive for NK-1, Ly-5, Ly-6, Ly-10, and AsGm-1 and Lyt-2 antigens. The Pre-NK or accessory cells are positive for Qa-5, Ly-10, and Ly-20 and partially positive for NK-1, Thy-1, and AsGm-1 antigens. These Qa-5+ NK cells do not exhibit cytotoxic activity to WEHI or P815. They could also be generated from BM of nude mice as well as beige mice. We concluded from these studies that rat Con A-conditioned medium contained factors that could differentiate Pre-NK cells to mature NK cells and that these cells are heterogeneous. This in vitro culture system is useful in delineating the ontogeny of NK cells.     

How do normal and leukemic white blood cells egress from the bone marrow? Morphological facts and biochemical riddles

Under normal circumstances only mature granulocytes and monocytes cross the bone marrow sinus wall, a trilaminar structure consisting of endothelial cells, a discontinuous basal membrane and an adventitial cell layer in order to get access to the blood circulation. In leukemia, however, immature white blood cells are able to traverse the barrier and to appear in the blood stream. Very little is known about the regulatory processes which govern the egress of white blood cells in healthy individuals and their malignant counterparts in patients with leukemia. The results of the few studies performed to address this question in animal and human leukemias all agree that the extent to which adventitial cells (fibroblasts) cover the endothelium in bone marrow is drastically reduced. This implies altered interactions between the leukemic and adventitial cells and their extracellular matrix. We propose here a model to explain the egress of normal cells and their leukemic counterparts. It is based upon our own experimental data and the general at present limited knowledge of the subject. It is hoped that this model will stimulate further research into this important aspect of leukemogenesis.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.     

Direct versus cultured preparation of bone marrow cells from 22 patients with acute myeloid leukemia

Summary The 24-h culture of bone marrow from patients with acute myeloblastic leukemia (AML) and acute promyelocytic leukemia (APL) gave more analyzable metaphase cells and improved chromosome morphology compared with direct preparations. Culture increased the proportion of cytogenetically abnormal cells, and in six bone marrows where the direct preparation failed, a result was obtained from the cultured preparation. The culture of bone marrow from patients with APL led to the detection of clones carrying the t(15;17) that were not found in direct preparations. Such sequestered clones were not found in AML and acute myelomonocytic leukemia (AMMoL). Cultured preparations were no better than direct preparations from AMMoL.     

小型猪骨髓间充质干细胞的分离和培养

目的建立小型猪骨髓间充质干细胞（mesenchymal stem cells,MSCs）的体外分离和培养方法。方法穿刺小型猪髂后上嵴抽取骨髓,经密度梯度法离心得到骨髓单个核细胞,接种后形成单层贴壁细胞。用形态学方法鉴定培养的MSCs。结果经培养存活的MSCs原代和一代呈纺锤型、多边型或星型。至二代起呈均一纺锤型,似成纤维细胞样,长宽比例约为（2～3）？1。体外培养的原代MSCs8～10d达到融合,传代后仍具有较强的增殖能力。结论小型猪MSCs可在体外长期、稳定培养,其分离、培养体系的建立为基础研究和组织工程技术提供了一个有价值的动物模型。     

DNA methylation analysis of bone marrow cells at diagnosis of acute lymphoblastic leukemia and at remission

To detect genes with CpG sites that display methylation patterns that are characteristic of acute lymphoblastic leukemia (ALL) cells, we compared the methylation patterns of cells taken at diagnosis from 20 patients with pediatric ALL to the methylation patterns in mononuclear cells from bone marrow of the same patients during remission and in non-leukemic control cells from bone marrow or blood. Using a custom-designed assay, we measured the methylation levels of 1,320 CpG sites in regulatory regions of 413 genes that were analyzed because they display allele-specific gene expression (ASE) in ALL cells. The rationale for our selection of CpG sites was that ASE could be the result of allele-specific methylation in the promoter regions of the genes. We found that the ALL cells had methylation profiles that allowed distinction between ALL cells and control cells. Using stringent criteria for calling differential methylation, we identified 28 CpG sites in 24 genes with recurrent differences in their methylation levels between ALL cells and control cells. Twenty of the differentially methylated genes were hypermethylated in the ALL cells, and as many as nine of them (AMICA1, CPNE7, CR1, DBC1, EYA4, LGALS8, RYR3, UQCRFS1, WDR35) have functions in cell signaling and/or apoptosis. The methylation levels of a subset of the genes were consistent with an inverse relationship with the mRNA expression levels in a large number of ALL cells from published data sets, supporting a potential biological effect of the methylation signatures and their application for diagnostic purposes.     

Impulse cytophotometric studies of bone marrow and blood cells in children with acute lymphoblastic leukemia (ALL). 1. Bone marrow cells

1. Compared with the peripheral blood lymphocytes of healthy children the cell fractions in the S- and G2 + M-phase are significantly higher in the bone-marrow of those children affected with ALL. This increase was proved in the SR- and MR-group irrespective of the cytomorphological subtype and cytochemical reaction. In patients with relapses the percentage of S-phase cells is below 6%. 2. In about 30% of our patients (mainly in the SR-group with L1-morphology) an initial DNA-aneuploidy was identified. As the risk of relapse is higher in children without DNA-aneuploidy, this symptom has a pretherapeutical-prognostic significance. 3. In the phase of hematological full remission, DNA-frequency distribution correlates with the proliferative activity of normal hematopoiesis. It provides no additional information about the pretherapeutical risk.     

IL-1 inhibits B cell differentiation in long term bone marrow cultures

There is evidence that stromal cells are responsive to changes in their external milieu and that this can affect their function. IL-1 has been identified as one mediator that can affect stromal cells by increasing their secretion of CSF. The monokine has also been reported to be a B cell differentiation factor. The purpose of this study was to test the effects of IL-1 on the pattern of hemopoietic cell differentiation by adding IL-1 alpha to myeloid long term bone marrow cultures (MBMC) at the time of their transfer to lymphoid bone marrow culture conditions. This usually results in the cessation of myelopoiesis and the induction of B lymphopoiesis. The addition of 50 U/ml of rIL-1 alpha, but not 10 U/ml, to MBMC at the time of their transfer to lymphoid conditions resulted in a complete inhibition of B cell differentiation and sustained myelopoiesis. To determine whether adherent layer cells contributed to this effect, conditioned medium (CM) was collected from adherent layers treated previously with the antibiotic mycophenolic acid. This depletes the hemopoietic cells from the cultures and retains a purified population of stromal cells. CM from mycophenolic acid- treated adherent layers exposed for 24 h to 50 U/ml of IL-1 was added at volume concentrations of 5, 10, and 25% to MBMC at the time of transfer to lymphoid bone marrow culture conditions and at each feeding thereafter. Expression of the B lineage associated 14.8 Ag and IgM was inhibited on a dose dependent basis, and myelopoiesis was sustained in cultures to which 25% CM had been added. Induction of B lymphopoiesis occurred in cultures to which adherent cell CM not exposed to IL-1 had been added. The CM from the IL-1-treated adherent cells contained CSF, because it promoted the growth of myeloid colonies from fresh marrow or MBMC cells and stimulated the granulocyte-macrophage-CSF sensitive FDC-P1 cell line to proliferate. IL-3 was not present in the CM, because stimulation of the IL-3 sensitive 32D cell line was not observed. The CM from the IL-1-treated adherent cells stimulated thymocytes to proliferate in the presence of PHA. This raised the possibility that the induced CSF may have required IL-1 to mediate their effects in the cultures. However, B lymphopoiesis was inhibited and myelopoiesis maintained upon addition of recombinant granulocyte-, macrophage-, and granulocyte-macrophage-CSF to cultures, indicating that IL-1 or other non-CSF molecules induced by it need not be present.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions between lymphocytes and hematopoietic progenitor cells in normal and leukemic human bone marrow (phase contrast observations)

Single cell observations of normal and of leukemic human bone marrow cells demonstrated cell-cell interactions of lymphocytes with hematopoietic progenitor cells. In all cases lymphocytes and target cells were from the same individual. Lymphocyte-target cell interactions occurred more frequently with normal committed progenitor cells and leukemic blast cells from acute myeloid leukemia than with precursor cells of the proliferative cell pool, including myeloblasts, promonocytes, erythroblasts and megakaryocytes. Both induction of mitosis and degeneration of the progenitor cells occurred after cell-cell interaction with almost the same frequency. Acute myeloid leukemic blast cells degenerated after contact with lymphocytes with the same frequency as normal progenitor cells (i. e. in 16% of cell contacts), but especially during mitosis. In contrast, normal and regenerating bone marrow progenitor cells from myeloproliferative diseases demonstrated no degeneration after cell-cell interaction with lymphocytes during mitosis. Otherwise the induction of mitoses by lymphocyte-target cell interactions was more frequently observed in normal progenitor cells than in leukemic blasts.