Flow cytometric determination of ploidy and proliferative activity on isolated bone marrow particles and on total bone marrow aspirate

The nuclear DNA content distribution of peripheral blood (PB) and bone marrow (BM) cells was determined by propidium iodide flow cytometry in 33 patients who underwent BM aspiration for diagnostic purposes. Two types of BM samples were taken during every aspiration procedure: whole BM aspirate, composed of BM particles contaminated by PB cells; isolated BM particles. Proliferative activity was calculated as the percentage of cells with DNA content intermediate between the diploid (2n) and the tetraploid (4n) values (2n-4n%). Ploidy was expressed as the ratio between the modal channel of the G0-G1 peak of the probe and that of an internal reference standard (DNA index, DI). The 2n-4n% was very close to zero in all PB samples. It was significantly greater in BM particles (21.2 +/- 6.6%) than in whole BM aspirate (16.6 +/- 5.5%, p less than .0005), with a close correlation (r2 = 66; p less than .0001) between the two values. Aneuploid stem lines were found in BM but not in PB. The DI of BM stem lines were similar in whole BM aspirate and BM particles, but the percentage of aneuploid cells was usually higher in BM particles. The reduced proliferative activity and the lower percent of aneuploid cells found in whole BM aspirates, with respect to BM particles, can be attributed to the contamination of BM tissue by PB, which had a very low proliferative activity and did not show aneuploidy. BM particles are therefore an easily obtained and reliable sample for routine evaluation of proliferative activity and ploidy of BM cells by DNA flow cytometry.     

Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells

Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells. The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.     

Suppressive effects of swainsonine and N-butyldeoxynojirimycin on human bone marrow neutrophil maturation

The effects of the N-linked oligosaccharide inhibitors swainsonine and N-butyldeoxynojirimycin (NB-DNJ) on granulopoiesis was investigated using human bone marrow cells in in vitro liquid and agar cultures. The addition of the inhibitors into cultures containing granulocyte colony-stimulating factor (G-CSF) suppressed maturation from myelocytes into mature neutrophils. Swainsonine did not induce apoptosis, but NB-DNJ induced considerable apoptosis, especially in the presence of G-CSF. This result indicated that the decrease of mature neutrophils by swainsonine was not because of cell degeneration. In the case of NB-DNJ, it was thought to be because of both maturation suppression and apoptosis. In a colony-forming unit-granuloid (CFU-G) colony assay, the number of colonies was increased in the presence of the inhibitors, but the morphology of colonies was predominantly compact, or immature. The inhibitors also suppressed the expressions of mRNAs of CCAAT/enhancer binding protein epsilon (C/EBPepsilon) and G-CSF receptor as markers of terminal neutrophil maturation. These findings suggested that the incompleteness of N-linked oligosaccharide leads to the suppression of terminal neutrophil maturation.     

Growth of human bone marrow in vitro

Attempts were made to establish human bone marrow (HBM) culture. Cell proliferation was triggered by medium conditioned by acute myelocytic leukemic (AML) cells exposed to 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A 6-15 fold multiplication of myelomonocytic cells was obtained in tissue culture from HBM cells. Proliferation of granulocyte-macrophage colony forming cells (CFC-GM) occurred up to 30-fold. The morphology of the cells and cytochemical markers were investigated at all stages of the culture.     

Rhythms in human bone marrow and blood cells

In 24h studies of bone marrow (BM), circadian stage-dependent variations were demonstrated in the proliferative activity of BM cells from subsets of 35 healthy diurnally active men. On an average, the percentage of total BM cells in deoxyribonucleic acid (DNA) synthesis phase was 188% greater at midday than at midnight (circadian rhythm: p = 0.018; acrophase or peak time of 13: 16h). Patients with malignant disease (n = 15) and a normal cortisol circadian rhythm showed higher fractions of BM cells in S-phase at midday. Colony-forming units--granulocyte/macrophage (CFU-GM), an indicator of myeloid progenitor cells, showed the same circadian variation as DNA S-phase (average range of change or ROC = 136%; circadian rhythm: p < 0.001; acrophase of 12:09h). Deoxyribonucleic acid S-phase and CFU-GM in BM both showed a circannual rhythm (p = 0.015 and 0.008) with an identical acrophase of August 12. The daily peak in BM glutathione content, a tripeptide involved in cellular defense against cytotoxic damage, preceded BM proliferative peaks by 4-5 h (ROC = 31-90%; circadian rhythm: p = 0.05; acrophase of 08:30h). Myeloid (ROC = 57%; circadian rhythm: p = 0.056; acrophase at 08:40h) and erythroid (ROC = 26%; circadian rhythm: p = 0.01; acrophase of 13:01h) precursor cells were positively correlated (r = 0.41; p < 0.001), indicating a circadian temporal relationship and equal influence on S-phase of total BM cells. Yield of positive selected CD34+ progenitor stem cells also showed significant circadian variation (ROC = 595%; circadian rhythm: p = 0.02; acrophase of 12:40h). Thus, the temporal synchrony in cell cycling renders BM cells more sensitive at specific times to hematopoietic growth factors and cell cycle-specific cytotoxic drugs. Moreover, proper timing of BM harvesting may improve progenitor cell yield. When using marker rhythms in the blood to allow for individualized timing of BM procedures, the times of low values in white blood corpuscles, neutrophils, and lymphocytes and high values in cortisol were predictive of the times of highest BM erythroid, myeloid, and total S-phase numbers occurring in the following 12 h.     

Feeder cells enhance oncolytic and proliferative activity of long-term human bone marrow interleukin-2 cultures and induce different lymphocyte subsets

Summary The effect of feeder cells on oncolytic activity of lymphocyte subsets and their growth was evaluated in long-term human bone marrow interleukin-2 (IL-2) cultures. Two B-lymphoblastoid cell lines (Daudi and Epstein-Barr-virus-transformed BSM) and two human leukemias, AML-M5, were used as feeder cells. The most prominent effects were seen in cultures stimulated with Daudi cells. In these cultures, cytotoxic activity was 100–1000 times increased against a broad range of target cells and the total cellular expansion was more than 40 times higher than in control cultures. This Daudi-related effect appeared to be mediated by natural killer (NK) cells, since cellular expansion occurred mostly in the CD16⁺ and CD56⁺ CD3^– NK cell subset. In cultures stimulated with BSM and acute myelogenous leukemia (AML) feeder cells, the increase in proliferation was similar, but the enhancement of cytotoxicity, even though significant, was less prominent. Although all feeder cells were effective in stimulation of bone marrow reactivity, the highest cytotoxicity was always observed with feeder cells autologous to the targets, indicating some degree of specificity. This was especially evident in cultures stimulated with autologous versus allogeneic AML feeder cells. In contrast to Daudistimulated IL-2 cultures, in which the highest expansion of CD3^– CD56⁺ NK cells was observed, in BSM and AML cultures, the CD3⁺ CD56^+/- T cell subsets were more prolific. This indicates that the response and phenotypic heterogeneity of bone marrow cultures depends on the type of feeder cells used. This observation indicates that the preferential stimulation of a pertinent lymphocyte subset for therapeutic purposes may be possible.Recipient of Florence Maude Thomas Cancer Research Professorship     

Lipid composition of human bone marrow

1. A modified method for the analysis of phospholipid mixtures by selective hydrolysis is described. 2. The phospholipid compositions of normal human bone marrow and of the bone marrows of patients who died with anaemia or various forms of leukaemia were investigated. 3. Phospholipids from normal bone marrow comprised about 44% of lecithin, 4% of choline plasmalogen, 7% of glyceryl ether phospholipid (choline base), 10% of sphingomyelin, 22% of phosphatidylethanolamine plus phosphatidylserine, 8% of ethanolamine plasmalogen and 5% of glyceryl ether phospholipid (ethanolamine base). 4. The proportion of kephalin (i.e. phosphatidylethanolamine plus phosphatidylserine) in the pathological bone marrows tended to be lower than normal. No other consistent differences were observed between the normal and pathological samples. 4. A ceramide dihexoside was isolated from normal bone marrow.     

The proliferative capacity of pure red cell aplasia bone marrow cells.

Pure red cell aplasia (PRCA) is a heterogeneous disorder. Immunologic abnormalities have recently been uncovered suggesting that both cell-mediated and humoral immune mechanisms may be of etiological importance in PRCA. Utilizing a technique for the cloning of bone marrow erythroid precursors, we determined the in vitro proliferative capacity of erythroid cells obtained from 21 patients with PRCA. Bone marrow cells from one group of patients produced normal or increased numbers of erythroid colonies while the in vitro proliferative capacity of bone marrow cells from a second group was characterized by subnormal erythroid colony formation. Sera obtained from the former group was frequently associated with demonstrable serum inhibitors of erythropoiesis, while PRCA in the latter group was probably the consequence of intrinsic erythroid stem cell defects or pathologic cellular interactions with nonerythroid regulatory cells. This survey of a relatively large population of patients with PRCA provides evidence for the multiple causative mechanisms that can be operative in the production of PRCA.     

Formation of bone tissue by mouse bone marrow cell suspensions in organ culture

Adult mouse bone marrow cell suspensions prepared by trypsinization were cultivated in gelatin sponges on millipore filters. When HAWP filters were used, multilayer bone structure was formed. It contained mineralized ground substance, incorporated bone cells and osteoblast layer. With the use of AUFS filters, bone tissue developed not only on the top surface, but also inside the filter.     

New series of lipoxins isolated from human eosinophils

Granulocytes from human eosinophilic donors were incubated with arachidonic acid or 15-hydroxyeicosatetraenoic acid (15-HETE) and stimulated with the ionophore A23187. The eicosanoids were extracted with reversed-phase cartridges and subjected to RP-HPLC analysis. When extracts from eosinophil-enriched populations were analysed and compared with extracts from human neutrophils, three additional peaks were detected which coeluted with 15-hydroxy-delta 13-trans-15H derivatives of leukotriene C4, D4 and E4 in different HPLC systems. The recorded absorbance spectra of the eluted compounds and the standards were identical and showed a maximum at 307 nm which is characteristic for a conjugated tetraene system with a bathochromic shift by the sulfur moiety in alpha-position to the tetraene system. The compound which coeluted with the 15-hydroxy-LTC4 standard was treated with gamma-glutamyltransferase and converted to the corresponding leukotriene D4 derivative. The results indicate that interaction between the 5- and 15-lipoxygenase pathways leads to the formation of a new series of arachidonic acid metabolites in human eosinophils. Since the biosynthetic route is similar to that of lipoxin A4 and lipoxin B4, we suggest the trivial names lipoxin C4, D4 and E4.