Analysis of human megakaryocytic cells using dual-color immunofluorescence labeling.

BACKGROUND: Megakaryocytes are classically identified by their cellular morphology and expression of platelet glycoproteins. METHODS: In this study, the expression of GPIIIa (CD61) on hemopoietic cells was analyzed by dual-fluorescence flow cytometry. RESULTS: All monocytic cells (CD14+) were shown to coexpress CD61. As the expression of platelet protein on these monocytic cells cannot be reduced by treating the cells with anticoagulant (ethlyenediaminetetraacetic acid [EDTA]), this observation is not simply due to platelet adhesion. When sorted CD61(lo)CD14+ cells were studied by light and electron microscopy, platelets or platelet fragments could not be detected on the cell surface. These cells were found to have typical monocytic morphology but no megakaryocytic characteristics. CONCLUSIONS: This finding demonstrates that without careful definition, the quantitation of megakaryocytic cells will be inappropriately high. A clear and unambiguous criteria for the identification of megakaryocytic cells is described based on the high expression of platelet glycoprotein (e.g., CD61(hi) or CD41(hi)) but not the monocyte marker (CD14(neg)).

Viable bone marrow stromal cells are required for the in vitro survival of B-cell precursor acute lymphoblastic leukemic cells.

The growth of B-cell precursor acute lymphoblastic leukemic (BCP ALL) cells in vitro is dependent on interactions with bone marrow (BM) stromal cells. We have recently demonstrated that the rate of cell division of BCP ALL cells increases when cultured in direct contact with BM stromal cells. A number of studies have examined the binding of BCP ALL cells to BM stromal cells and extracellular matrix components. To date there have been no studies examining the effect of such binding on the growth and survival of BCP ALL cells. In this study, by measuring the growth parameters of these cells with use of a lipophilic fluorescent probe, PKH 26 GL, we demonstrate the positive effect of viable BM stromal cells on BCP ALL cell survival in 10 patient samples. At the same time, by comparing these cultures with cultures of the same patient samples in the presence of glutaraldehyde-fixed stromal cells, deoxycholic acid-derived stromal cell matrices, purified laminin, collagen or fibronectin, the role of various stromal cell-derived contact components in BCP ALL survival was tested. It was shown that the survival of BCP ALL cells in vitro was dependent upon viable BM stromal cells present in co-culture as the various contact components did not show any functional effect on BCP ALL cell survival.

Flow cytometric analysis of intercellular adhesion between B-cell precursor acute lymphoblastic leukemic cells and bone marrow stromal cells.

The growth of B-cell precursor acute lymphoblastic leukemic (BCP ALL) cells in vitro is dependent on interactions with bone marrow (BM) stromal cells. We have recently demonstrated that the rate of cell division of BCP ALL cells increases when cultured in direct contact with BM stromal cells. In this study we describe a new method for examining the direct binding of BM stromal cells and BCP ALL cells at a cellular level. For this binding assay, BCP ALL cells from six patient samples were first stained with the lipophilic fluorescent probe PKH 26 GL and mixed with BM stromal cells in suspension. In all cases, aggregates between BCP ALL and BM stromal cells were identified by flow cytometry and isolated. Using this assay we have examined some of the mechanisms involved in this binding process. The pattern of aggregate formation at various leukemic/stromal cell ratios showed that the aggregate formation increased by increasing the number of either cell type and that the binding could not be saturated. This suggests that the interaction between these cells is an equilibrium reaction. Functional studies showed that the majority of BCP ALL-BM stromal cell binding is dependent on the presence of divalent cations and requires active cellular metabolism. Finally, by use of inhibitory monoclonal antibodies (moAbs) directed against cell adhesion molecules including anti-CD29, VCAM and CD18, we have demonstrated that the involvement of these molecules in the direct cellular interactions could be detected by this method. However, the maximum inhibition observed was 36% which suggests either that the avidity is low or that other adhesion molecules are involved. The data show that the use of flow cytometric analysis of aggregate formation (rather than cell binding to intact cell layers) allows the study of cell interactions at the individual cell level which can reveal additional cellular adhesion mechanisms.

Human bone marrow stromal cell contact and soluble factors have different effects on the survival and proliferation of paediatric B-lineage acute lymphoblastic leukaemic blasts.

Recent studies have confirmed that in vitro viability and proliferation of precursor B-cell leukaemia (ALL) cells are linked to the presence of bone marrow derived stromal cells. To investigate whether this effect is mediated by direct contact or through the action of soluble factors, using a method we have recently described, the growth parameters of ALL bone marrow blast cells from eight newly diagnosed patients were determined with the lipophilic fluorescent probe PKH 26 GL. The viability of ALL cells and the rate of cell division in cultures containing either medium alone; stromal cell conditioned medium; stromal cell layers allowing direct contact, or in 0.4 microns microporous membrane cultures suspended above stromal cell layers were examined. In all eight samples an improved maintenance of ALL cells in a viable state in cultures containing bone marrow stromal cells was observed. The survival of leukaemic cells was equivalent in 0.4 microns microporous membrane cultures suspended above stromal cell layers and in cultures of leukaemic cells in direct contact with stromal cell layers. It was thus demonstrated that this effect was mediated by the action of soluble factor(s) present in these cultures. However, the improved maintenance of ALL cells in a viable state was observed in only one of the eight cases when ALL cells were cultured in stromal cell conditioned medium alone. The highest rate of cell division of leukaemic cells was observed in ALL cells in direct contact with bone marrow stromal cells. The activities of stromal cell derived soluble factors could not be reproduced by recombinant forms of likely candidate factors including IL-1 beta, IL-4, IL-6, IL-7, SCF, TNF alpha, TGF beta, LIF, NGF or a mixture of these factors when examined in cultures of the same patient samples. This study implicates the existence of a novel bone marrow derived factor(s) that improves survival of ALL cells in vitro.

Measurement of the growth parameters of precursor B-acute lymphoblastic leukaemic cells in co-culture with bone marrow stromal cells; detection of two cd10 positive populations with different proliferative capacities and survival.

A new assay system using the fluorescent probe PKH 26 GL was employed to investigate the regulation of precursor B-cell acute lymphoblastic leukaemic (ALL) cell growth. PKH 26 GL is a lipophilic fluorescent probe which becomes incorporated into the plasma membrane upon the staining of cells. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. Bone marrow derived ALL cells from seven newly diagnosed cases were stained with PKH 26 GL, and cultured for 14 days in control cultures without stimulus, or in cultures with preformed human bone marrow stromal cell layers. Viable leukaemic cells from these cultures were identified on the basis of forward light scatter, 90 degrees light scatter, propidium iodide exclusion, PKH 26 GL staining and CD10 expression by flow cytometry at the beginning of the culture period and on days 2, 6, 10 and 14. The growth parameters of these leukaemic cells were determined by analysis of their pattern of PKH 26 GL fluorescence. A higher rate of proliferation and survival of cells was observed in cultures with stromal cells compared with control cultures, without stromal cells. In the presence of stromal cells, survival and proliferation continued throughout the culture period; in contrast in five of seven control cultures no viable cells could be detected after 6-10 days. Interestingly, two populations of leukaemic cells were distinguished on the basis of their different rates of proliferation, when co-cultured with stromal cells. The results indicate that this technique provides a means for studying and quantitating leukaemic cell growth within a complex stroma-dependent system.

A novel approach to the measurement of different in vitro leukaemic cell growth parameters: the use of PKH GL fluorescent probes.

The application of the fluorescent cell membrane probes PKH2 and PKH 26 GL in the measurement of leukaemic cell growth was examined on four cell lines K562, NALM-6, ACV (a pre-B cell line) and HL-60 using flow cytometry. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. By measuring the mean fluorescence intensity of the cells at the beginning of culture and at various time points, and by combining this information with a viable cell count, it was possible to determine: (1) the number of viable cells; (2) their rate of proliferation; (3) their number of cell divisions; and (4) the maintenance of cells in a viable state over a period of time. It was demonstrated that these parameters could be reliably established using the red fluorescent probe PKH26 GL. In contrast, the green fluorescent probe PKH2 GL showed dye transfer resulting in an underestimation of the number of cell divisions and an overestimation of the maintenance of cells in a viable state. The potential advantages of the use of PKH26 GL over conventional assays for the measurement of leukaemic cell growth parameters are discussed.

Simple immuno bead labeling of leukemic cell clusters cultured in methylcellulose.

In studies of in vitro leukemic clonogenic cells it is of importance to determine the cell lineage of individual clusters grown in culture. A method is described for the in-situ identification of leukemic cell clusters in methylcellulose cultures. Whole cultures are dried and incubated with various monoclonal antibodies, followed by incubation with beads coated with secondary antibody. Clusters containing antibody-positive cells are heavily labeled with beads which simplifies the recognition and scoring of clusters using normal light microscopy. This method has general applications and can also be used to identify normal myeloid and lymphoid clusters depending on the availability of lineage specific monoclonal antibodies.

Selection of two human bone marrow stromal cell subpopulations with different effects on the maintenance and differentiation of myeloid progenitor cells.

The simple selection of two human bone marrow stromal cell populations is described. Adherent stromal cell layers were formed in primary cultures of low-density marrow cells. At time of confluence, persistent nonadherent cells were collected and transferred to new culture flasks, where they formed a secondary stromal layer. These primary and secondary stromal layers differed in their ability to support myelopoiesis, as tested by progenitor cell production after inoculation with fresh bone marrow cells. In the presence of primary stromal layers the number of granulocyte-macrophage colony-forming cells (GM-CFC) decreased gradually, but in the presence of secondary layers production of GM-CFC was evident during the first 3 weeks. The regulation of the two stromal types on hemopoietic cell proliferation and differentiation was investigated by determining the kinetics of the transitions within the differentiation sequence of three myeloid progenitor cells. Pre-CFC, day-14 CFC, and day-7 CFC were fractionated by cell sorting on the basis of forward light scatter and cocultured with the two stromal layer types. It was found that the decrease in CFC numbers in the presence of primary stromal layers could be explained by the stimulation of hemopoietic cells into rapid differentiation with loss of proliferative capacity at an early stage of culture. Secondary layers appeared to promote survival and self-renewal of later types of progenitor cells and to trigger more immature cells to proliferate and differentiate at a later time of culture.

Cellular cytotoxicity assessed by the 51Cr release assay. Biological interpretation of mathematical parameters.

This study deals with the interpretation of primary data of the 51Cr release assay for cellular cytotoxicity. In particular the dose-effect relationship between increasing numbers of lymphoid cells and the percentage of target cells killed has been considered. The number of target cells killed depends on the frequency and the activity of cytotoxic cells. These two parameters are often not distinguished from each other, which causes confusion and frequently results in vague descriptions of cytotoxicity data. Because in many cases not all cells in the target cell population can be lysed, we recommend the introduction of the plateau value for target cell kill. This maximum of target cell kill is a measure of the frequency of lysable target cells, but also depends on the cytotoxic cell activity. Description of the dose-effect curve by y = A(1 - e-kx) allows the simultaneous calculation of the maximum kill (A) and the frequency of cytotoxic cells (k) in the lymphoid cell population (x). Results are presented which indicate that A and k represent totally independent biological parameters the use of which permits a more objective description of cytotoxicity data.

Rapid expansion of human cytotoxic T cell clones: growth promotion by a heat-labile serum component and by various types of feeder cells.

Culture conditions are described that result in rapid expansion of cloned cytotoxic T cells of human origin. A combination of allogeneic lymphocytes and Epstein-Barr virus (EBV) transformed B cells (B-LCL) as irradiated feeder cells resulted in a 10-fold higher cell yield than obtained by use of either feeder cell alone. The large cell numbers obtained in a relatively short period of time facilitate in vitro and in vivo experimentation. Further enhancement of cell proliferation was obtained by the use of fresh human serum not heat-inactivated before use. This suggests the presence of a heat-labile growth stimulating factor or factors in human serum. Round-bottomed microtitre wells were found to give best culture results. Plating and harvesting of cells cultured in wells was facilitated by a specially designed culture flask. Addition of leucoagglutinin (purified phytohaemagglutinin) to the culture medium resulted in an approximately 3-fold higher cell yield. Optimal culture results were obtained when all the above factors were combined. It was possible to expand single cytotoxic T cells to up to 10(9) cells in about 30 days with full retention of cytolytic activity and target cell specificity. T cell clones have now been cultured for more than 70 generations.

Differentiation restriction in the neutrophil-granulocyte, macrophage, eosinophil-granulocyte pathway: analysis by equilibrium density centrifugation.

Bone marrow culture techniques and equilibrium density centrifugation of human bone marrow cells were used to analyse the neutrophil-granulocyte, macrophage and eosinophil-granulocyte progenitor hierarchy. The buoyant density of progenitor cells changes as cells differentiate down the granulocyte-macrophage pathway and this allows the construction of a density 'map' of the points at which differentiation decisions are made. Unipotent progenitors, neutrophil-granulocyte (G), monocyte-macrophage (M), eosinophil-granulocyte (Eo), are more dense than bi- and tripotent progenitors (GM and EoGM) and have a lower 7-day proliferative capacity (assessed as the clone size achieved in maximally stimulated agar cultures). Experiments in which marrow cells were separated on a basis of their density and either cultured in agar immediately or after an interval of 6 days in suspension culture, were performed to establish the density of the cells which give rise to each type of progenitor, i.e. to investigate parent-progeny relationships. In each case the parent cells were of lower density than the unipotent or bipotent progenitor in question. The ability to separate, at least partially, unipotent, bipotent and multipotent cells of closely related lineages is important since it facilitates studies of the intracellular events taking place as restriction of the cell's differentiation options takes place.

Physical and kinetic properties of haemopoietic progenitor cell populations from mouse marrow detected in five different assay systems.

Properties of haemopoietic progenitor cells detected in several different assays have been compared in order to position them within the haemopoietic developmental lineage. The spleen colony-forming cell (CFUs), the high proliferation potential colony-forming cell (HPP-CFC) and two granulocyte-macrophage colony-forming cells (GM-CFC-1 and GM-CFC-2) have been studied. Two experimental techniques were used: separation of cells on the basis of their buoyant density and comparison of the survival of haemopoietic cells after donor mice had been injected with the cytotoxic drug 5-fluorouracil (5-FU). On linear BSA gradients the modal buoyant densities of CFUs, HPP-CFC and GM-CFC-1 were the same, 1.070 g cm-3; the density of GM-CFC-2 was higher, 1.075 g cm-3. GM-CFC-2 colonies were much smaller and contained far fewer cells than HPP-CFC or GM-CFC-1 colonies, even after prolonged culture, and this suggests that dense haemopoietic progenitors have less proliferation potential. This was confirmed by comparison of the size of colony formed, under identical culture conditions, by progenitors of different densities. Mean colony diameter was inversely related to the density of the progenitor cell. With the exception of GM-CFC-1, low density progenitors were more resistant to the cytotoxic effects of 5-FU than high density precursor cells (GM-CFC-2). Consequently, the GM-CFC-1 could be distinguished from GM-CFC-2 on the basis of buoyant density and from the other low density populations on the basis of post-FU kinetics. The reasons why the GM-CFC-1 should be more sensitive to 5-FU than other low density progenitors are discussed and the relation of these low density precursors to one another in terms of their position within the haemopoietic developmental lineage is elucidated.

A two-step procedure for obtaining 80-fold enriched suspensions of murine pluripotent hemopoietic stem cells.

A method to enrich for murine pluripotent hemopoietic stem cells is described. It consists of two steps. Cells with a density of less than 1.072 g/cm3 at 4 degrees C are first selected from mouse bone marrow by a single-step bovine serum albumin density separation. These low-density cells are then labeled with fluoresceinated wheat germ lectin and sorted by a light-activated cell sorter on the basis of differences in light scatter and fluorescence intensities. Numbers of pluripotent stem cells were determined by the spleen colony assaY (CFU-S). The concentration of CFU-S was 80 times higher in the sorted cell suspension than in the unfractionated control.

Characterization and enrichment of murine hemopoietic stem cells by fluorescence activated cell sorting.

Several methods for supravital staining of mouse bone marrow cells are described. These methods were employed to sort fractions of bone marrow cells by a light activated cell sorter and to determine cytochemical properties of hemopoietic stem cells. Labelling with fluoresceinated wheat germ lectin confirmed that the stem cells are among the bone marrow cells with the highest surface density of N-acetylneuraminic acid. Staining with tetracycline revealed that the stem cells contain relatively active or relatively many mitochondria. Staining with the bisbenzimide H33342, a supravital DNA stain, indicated that most pluripotent stem cells are in a G0/G1 state of the cell cycle. Using these and other measurements, separation protocols can be devised to concentrate stem cells. Some of these are discussed.

Clone size potential and sensitivity to colony-stimulating activity: differentiation-linked properties of granulocyte-macrophage progenitor cells.

Previous studies using semi-solid agar cultures of acute myeloid leukaemia (AML) cells have shown that proliferative capacity (clone size potential) and the degree of sensitivity of clone-forming cells to the specific granulocyte-macrophage regulatory colony-stimulating activity (CSA) are closely linked AML cell properties. The purpose of this study was to determine whether this association is confined to AML cells or whether the linking of these two properties in AML represents retention of an association occurring in normal granulocyte-monocyte progenitor cell populations. Bone marrow cells from normal donors were studied using four independent techniques to enrich for clonogenic cells with different clone size potentials (equilibrium density centrifugation, adherence to microcarrier beads, osmotic lysis and fluorescence-activated cell sorting). It was shown that mean clone size was directly related to the mean CSA threshold (amount of CSA required to stimulate 50% of the cells to clone formation). Further studies (including analysis of the kinetics of production of clonogenic cells with different clone size potentials in suspension culture) suggested that these two properties were linked to differentiation, with proliferative capacity decreasing and sensitivity to CSA increasing as cells differentiate down the granulocyte-monocyte pathway.

Induction of erythropoietin responsiveness in vitro by a distinct population of bone marrow cells.

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid 'burst-forming units' (BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.

Structural identity of the pluripotential hemopoietic stem cell.

A review is presented of the experiments that resulted in the identification of a specific morphologic entity representing the pluripotential hemopoietic stem cell (HSC) in mouse bone marrow. This entity was subsequently discovered in concentrated HSC preparations from bone marrow of rats, monkeys, and humans. In the mouse, a set of physical parameters (of the HSC) has been collected which agree with its morphologic description. It was also shown that these physical properties, and a number of cell surface properties, do not enable a distinction between HSC and its immediate descendants, the G/M CFU 1 and the E-BFU. The factors that stimulate proliferation of these three cell types have been isolated from human leukocyte conditioned medium and mouse spleen conditioned medium and were partly purified and characterized. The information at present indicates that the three cell types respond to closely related, if not identical, factors. Direct counts of HSC in electron microscopic preparations of density gradient fractions of different enrichment have been compared with HSC values computed from spleen colony counts and f factors for rat and mouse marrow. A high degree of correlation was found between the two types of observations. The slopes of the regression lines for mouse marrow fractions, for concentrates of normal rat marrow, and for concentrates of cycling rat marrow were the same, namely, 0.5. The deviation of this value from the expected value of 1.0 is probably not due to the use of erroneous f values. It is proposed that the observed discrepancy may be due to heterogeneity of spleen colony forming cells, in that a proportion of them may not be pluripotential.