Stem cell maintenance and commitment to differentiation in long-term cultures of murine marrow obtained from different spatial locations in the femur

Abstract. Murine bone marrow was separated into axial and marginal fractions in order to investigate the ability of cells from different spatial locations in the marrow to establish long-term cultures. The maintenance of haemopoiesis was significantly poor in long-term cultures of marginal marrow compared with axial or control (unfractionated marrow) cultures. Using techniques to further fractionate the axial or marginal marrow by depleting either stromal or haemopoietic cells, it was possible to investigate the relative importance of stromal and haemopoietic cell components. In the combinations studied, the more important determinant of effective in vitro haemopoiesis was the source of the haemopoietic cells rather than the stroma. The most effective stem cell maintenance and commitment to differentiation was observed when the source of the haemopoietic population was axial marrow. The data are consistent with axial marrow being a source of 'high quality' stem cells and this quality being an intrinsic property of the cells rather than one imposed by the stromal environment.     

Osteocalcin promotes differentiation of osteoclast progenitors from murine long-term bone marrow cultures

Murine long-term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM-CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either α-MEM with 2% heat-inactivated horse serum alone (α) or supplemented with 10% L-cell-conditioned medium (as a source of M-CSF) (L10). Few substrate-attached cells survived in basal α medium, but when treated with L10 medium or GM-CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM-CSF had large numbers of multinucleated cells, more than half of which were tartrate-resistant acid phosphatase–positive (TRAP). Osteocalcin further promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 μg/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate-resistant acid phosphatase activity in the osteocalcin plus GM-CSF group (75.6 ± 14.2) than in GM-CSF alone (53.3 ± 7.3). In the absence of M-CSF, GM-CSF stimulated tartrate-resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal-free subpopulation of hematopoietic progenitors in the presence of GM-CSF and L-cell-conditioned medium. These results are consistent with the hypothesis that this bone-matrix constituent plays a role in bone resorption. © 1994 Wiley-Liss, Inc.     

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.     

Rate of non-adherent cell loss from long-term cultures of murine bone marrow: effect of medium conditioned by the adherent cell population

The rate of random spontaneous cell loss from the non-adherent cell population of long-term cultures of murine bone marrow was determined. The measured rate of non-adherent cell loss is affected by previous conditioning of the medium in which the non-adherent cells are suspended. Specifically, the measured rate of non-adherent cell loss is significantly slowed when the non-adherent cells are suspended in medium conditioned in long-term haematopoietic cultures instead of fresh medium. It appears that a subset of the adherent cell population is the source of the factor or factors within the medium which result in this slower measured rate of non-adherent cell loss. This effect may be due to the stimulation to division of haematopoietic progenitor cells, offsetting the lysis of other non-adherent cells.     

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.     

In vitro differentiation of B lymphocytes from primitive hemopoietic precursors present in long-term bone marrow cultures

B lymphocytes are not produced in the Dexter long-term bone marrow cultures, but a primitive B cell precursor is present. The findings presented in this study demonstrate that this precursor can be induced to produce B lymphocytes by transferring the cultures to the Whitlock conditions for the long-term growth of B cells in vitro. Two weeks after the transfer of cultures maintained at 33 degrees C in medium supplemented with horse serum and steroids to low concentrations of fetal calf serum at 37 degrees C, marked effects can be observed. The pattern of cell growth changes from one in which the hemopoietic cells are clustered in tight foci containing several hundred cells to smaller ones in which the cells are not as densely packed. Fat cells in the adherent layer disappear and the supporting stroma becomes more uniform in appearance. This change in the culture format is accompanied by a decrease in the number of nonadherent cells and a shift from myelopoiesis to lymphopoiesis. The numbers of granulocyte-macrophage progenitors decline weekly after the change in culture conditions and are not detected by the third week. B cell colony-forming units appear by 3 wk. Cells that express the 14.8 cell surface antigen are induced by 1 wk after the change in culture conditions, followed by the appearance of surface IgM-bearing cells 2 wk later. This shift to lymphopoiesis can be confirmed morphologically. Granulocytes and macrophages disappear from the cultures by 4 wk, at which time almost all of the cells have a characteristic lymphocyte morphology. Upon switching these cultures back to the original Dexter conditions, only low levels of transient myelopoiesis can be reinitiated.     

Induction and long-term maintenance of Thy-1 positive T lymphocytes: Derivation from continuous bone marrow cultures

In the present study we investigated the presence of T-lymphocyte progenitors in the long-term murine bone marrow culture system described by Dexter: mature Thy-1 antigen-bearing T lymphocytes are lost in these cultures after a few days. By culturing nonadherent cells from such cultures in the presence of a supernatant of concanavalin A-stimulated spleen cells, a source of T-cell growth factor, we found that Thy-1 positive blast cells proliferated together with a second population of Thy-1 negative cells. These two populations of cells have been maintained in long-term in vitro cultures by passaging the cells in fresh conditioned medium at regular intervals. Moreover, we have been able to establish pure cultures of the Thy-1-bearing blast cells after separating them from the non-T cells using their adherence property to plastic surfaces. Long-term cultures of T lymphocytes can thus be established from long-term marrow cultures as well as from the spleen, thymus or fresh bone marrow.     

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.     

Seric factors influence cell cycle modifications during commitment of murine erythroleukemia cells to differentiation.

Cell cycle modifications are among the early events which take place during the induced differentiation of murine erythroleukemia (MEL) cells; a transient accumulation of the cells in the G1 phase of the cell cycle, followed by a re-entry of the cells into a proliferation state, has been described. In order to characterize a putative role of serum in such variations, we have studied the modifications of the cell cycle parameters when cells were induced to differentiate in the presence or in the absence of seric factors. We show that, in the absence of exogenous factors brought by serum, the G1 accumulation was enhanced both in amplitude and in duration, but cells were still able to bypass the G1 block and re-enter into the S phase. These results indicate that the resumption of cell proliferation after the transient block is under synergistic control of seric and endogenous factors, but these later are sufficient to overcome the block. However, MEL cells were unable to differentiate in the absence of seric factors, as measured by the number of benzidine-positive cells during induction with hexamethylene-bisacetamide (HMBA) or butyric acid. This capacity to differentiate was recovered when serum was added back to the culture medium, and the efficiency of recovery was maximal when cells underwent a full round of DNA replication in the presence of serum after the G1 block. The analysis of two molecular markers of cell differentiation confirmed these results.     

Spectral characteristics of caries autofluorescence obtained from different locations and caries severities

Dental caries usually occurs at interproximal and occlusal surfaces. The purpose of the present study was to determine if characteristic spectral factors extracted from autofluorescence (AF) spectra are informative regarding caries detection and the determination of caries stage as compared with DIAGNOdent results. AF spectra were obtained from caries lesions of different severities at two locations using a 405 nm laser. Three spectral factors, that is, spectral slope at 550 to 600 nm, spectral area under the curve at 500 to 590 nm and two‐peak ratio between 625 and 667 nm, were extracted. The values of three spectral factors linearly decreased as caries progressed. According to micro‐CT images, conventional visual and tactile inspections of lesions under or overestimated (25%‐65%) caries states, and brown or thickly stained layer on interproximal or occlusal surfaces, respectively, caused misclassifications of caries stage. Of the spectral factors examined, spectral slope and area under curve for interproximal and occlusal surfaces, respectively, were found to be significantly related to caries stage and showed least data overlap. For interproximal and occlusal surfaces, DIAGNOdent readings of different stages overlapped considerably though their mean values were significantly different regardless of stage.      

Inhibition of hemopoiesis in murine marrow cell cultures by recombinant murine tumor necrosis factor alpha: evidence for long-term effects on stromal cells

The addition of recombinant murine tumor necrosis factor alpha (rmTNF-alpha) to serum-free methylcellulose cultures inhibited macrophage colony formation stimulated by purified colony stimulating factor-1 (CSF-1), recombinant granulocyte-macrophage-CSF (rmGM-CSF), and recombinant interleukin 3 (rmIl-3). The concentration of rmTNF-alpha inhibiting colony formation by 50% (IC50) was between 2 and 20 ng/ml. Erythroid colony formation in cultures with erythropoietin (EPO) alone or EPO, rmIl-3, and rmGM-CSF in combination were reduced to a much lesser extent. In established long-term marrow cultures (LTMC), addition of 20 and 200 ng/ml of rmTNF-alpha resulted in release of cells from the adherent layer during the first week. Treatment of cultures with rmTNF-alpha for 4 consecutive weeks led to prolonged inhibition of cell production lasting up to 8 weeks after cessation of treatment. One day after addition of a low dose of TNF (2 ng/ml), "fat" cells were no longer observed in the adherent layer. Our results indicate that TNF inhibition of hemopoiesis occurs both at the progenitor cell and stromal cell levels.     

The effect of mouse lung granulocyte-macrophage colony-stimulating factor and other colony-stimulating activities on the proliferation and differentiation of murine bone marrow cells in long-term cultures

The roles of colony-stimulating factors in long-term bone marrow cultures were studied and compared. After single additions of high concentrations of unpurified colony-stimulating activities to the cultures, rapid deterioration of the cultures was observed. This appears to result from contaminants in the stimulatory preparations. Cultures to which one purified colony-stimulating factor (CSF) from endotoxin mouse lung-conditioned medium was added did not run down ten weeks after addition and were found to be the same as the controls. The deterioration of the cultures to which unpurified stimulators were added could not be accounted for by accelerated granulopoiesis leading to subsequent exhaustion of the cultures. The inability of purified CSF to affect the cellularity of the suspension cells did not result from instability or masking of the activity in the cultures, nor did CSF preferentially stimulate the cells within the adherent layer. The suspension cells responded to purified CSF after separation from the adherent cells. The data suggest that if CSFs are marrow stimulators, their effects in turn may be stringently regulated within the marrow.     

Morphological and functional differentiation in epithelial cultures obtained from human skin explants

The present study was undertaken to characterize primary epithelial cultures obtained from human skin explants as experimental systems for studies of the differentiation process. When human skin explants were incubated at 34-35 degrees C, fibroblastic growth was strongly inhibited, whereas the epithelial growth proceeded unchanged. The lateral growth of the epithelial cells could be divided into two phases - a migratory and a proliferative one. Only cultures incubated at 35 degrees C or below completed the morphological differentiation process before sloughing, whereas no qualitative difference in protein synthesis was observed between cultures incubated at temperatures from 33-37 degrees C. Cultured epidermal cells were labelled with 3H-thymidine and analysed by flow cytometry and cell sorting. Cells sorted from the S- and G2-phase populations were further analysed by autoradiography and a considerable heterogeneity as to the nuclear labelling was disclosed. A large fraction of S-phase cells were found to be totally unlabelled. The grain count distributions revealed similar cell cycle subpopulations as have been shown to occur in vivo. The relationship of these subpopulations to the differentiation process is discussed.     

Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures.

In a series of studies, we have reported that 1,25-dihydroxyvitamin D (3), a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D (3) and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D (3) decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1alpha, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D (3), but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D (3). Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D (3)-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D (3) on proliferating cells.     

Biology of marrow stromal cell lines derived from long-term bone marrow cultures of Trp53-deficient mice.

To investigate the effect of Trp53 (formerly known as p53) on stromal cells of the hematopoietic microenvironment, long-term bone marrow cultures were established from mice in which the Trp53 gene had been inactivated by homologous recombination (Trp53(-/-)) or their wild-type littermates (Trp53(+/+)). Long-term bone marrow cultures from Trp53(-/-) mice continued to produce nonadherent cells for 22 weeks, while Trp53(+/+) cultures ceased production after 15 weeks. There was a significant increase in the number of nonadherent cells produced in Trp53(-/-) long-term bone marrow cultures beginning at week 9 and continuing to week 22 (P < 0.02). The Trp53(-/-) cultures also showed significantly increased cobblestone island formation indicative of early hematopoietic stem cell-containing colonies beginning at week 10 (P < 0.01). Cobblestone islands persisted until weeks 15 and 22 in Trp53(+/+) and Trp53(-/-) cultures, respectively. Co-cultivation experiments in which Trp53(+/+) Sca1(+)lin- enriched hematopoietic stem cells were plated on Trp53(-/-) stromal cells showed increased cobblestone island formation compared to Trp53(-/-) Scal+lin- cells plated on Trp53(+/+) or Trp53(-/-) stromal cells. Radiation survival curves for clonal bone marrow stromal cells revealed a similar D0 for the Trp53(+/+) and Trp53(-/-) cell lines (1.62 +/- 0.16 and 1.49 +/- 0. 08 Gy, respectively; P = 0.408), and similar n (8.60 +/- 3.23 and 10.71 +/- 0.78, respectively) (P = 0.491). Cell cycle analysis demonstrated a G2/M-phase arrest that occurred 6 h after irradiation for both Trp53(+/+) and Trp53(-/-) stromal cell lines. After 10 Gy irradiation, there was no significant increase in the frequency of apoptosis detected in Trp53(+/+) compared to Trp53(-/-) marrow stromal cell lines. In the stromal cell lines, ICAM-1 was constitutively expressed on Trp53(+/+) but not Trp53(-/-) cells; however, a 24-h exposure to TNF-alpha induced detectable ICAM-1 on Trp53(-/-) cells and increased expression on Trp53(+/+) cells. To test the effect of Trp53 on the radiation biology of hematopoietic progenitor cells, the 32D cl 3 cell line was compared with a subclone in which expression of an E6 inserted transgene accelerates ubiquitin-dependent degradation of Trp53, thus preventing accumulation of Trp53 after genotoxic stress. The radiation survival curves were similar with no significant difference in the D0 or n, or in the percentage of cells undergoing apoptosis after 10 Gy irradiation between the two cell lines. Cells of the 32D-E6 cell line displayed a G2/M-phase arrest 6 h after 10 Gy, while cells of the parent line exhibited both a G2/M-phase arrest and a G1-phase arrest at 24 and 48 h. The results suggest a complex mechanism of action of Trp53 on the interactions between stromal and hematopoietic cells in long-term bone marrow cultures.     

In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes

Background  

Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species) were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established.     

Probability that the commitment of murine erythroleukemia cell differentiation is determined by the c-myc level

During the commitment of mouse erythroleukemia cell differentiation, c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the commitment of these cells, we have introduced the rat c-myc gene driven by inducible, heterologous (human metallothionein IIA) gene promoter into murine erythroleukemia cells and we have examined the ability of the transformed cells to undergo commitment to terminal differentiation. The induction of the exogenous c-myc gene expression inhibited the commitment of these cells. Time-dependent inhibition of the commitment was observed with the addition of zinc at an appropriate time after the induction with dimethyl sulfoxide. The result clearly indicated that late decline, not early decline, is required for the commitment. By examining the transformants expressing the exogenous c-myc mRNA at different levels, and the induction of the exogenous c-myc mRNA by varying the concentration of zinc, we demonstrated that the commitment may be determined by a stoichiometric amount of c-myc in the defined period. The data also suggest that the probability value for the commitment process occurring in a stochastic manner is well-correlated with the amount of c-myc mRNA.     

Comparative study of murine B-cells obtained by 2 different methods proliferating long-term in vitro

The hybridization of myeloma cells NP with lymphocytes of mice, immunized with protein isolated from Neisseria meningitidis strain Bc5 in a single injection into the spleen 3 days prior to fusion, made it possible to obtain 25-72% of hybridomas secreting antibodies to meningococcal antigens. The treatment of immune lymphocytes from these mice with the total preparations of nucleic acids, isolated by the phenol-detergent method from mouse myeloma cells NP and NS/0, induced an increase in the proliferative activity of lymphocytes; in some microcultures multilayer cell growth was observed on the bottom of the wells, whereas in the control microcultures such growth was absent. No synthesis of specific antibodies was detected in the cultures of lymphocytes whose proliferation was stimulated with nucleic acids.