Mitogen-stimulated Spleen Cell Conditioned Medium Research Articles

A self-renewing, bipotential erythroid/mast cell progenitor in continuous cultures of normal murine bone marrow.

We have established permanent lines of nonadherent cells from fresh normal mouse bone marrow in media containing pokeweed mitogen-stimulated spleen cell conditioned medium (PWSCM). These lines continuously produced erythropoietic progenitor cells (detected by their ability to form erythroid bursts in semi-solid medium containing erythropoietin) together with cells having characteristics of the mast cell lineage (as demonstrated by metachromatic staining with toluidine blue, histamine content and membrane receptors for IgE). Sixteen such cell lines have been established in sixteen attempts. Cloning experiments were carried out to determine the nature of the progenitor cell(s) responsible for the permanence of these cultures. When cells were cultured in methylcellulose medium containing PWSCM, colonies were observed which reached macroscopic size after 4 weeks of incubation. Replating of individual primary colonies resulted in secondary colony formation, indicating the presence of progenitor cells with self-renewal potential. Forty-seven primary colonies were picked and their cells were suspended in liquid culture medium containing PWSCM. Of these, twenty-one could be expanded to establish permanently growing sublines. Sixteen of these sublines were found to be composed of both erythroid progenitors and mast cells. In five sublines only mast cells could be seen; none of the sublines appeared to be purely erythroid. Karyotypic analysis of mast cells and of erythroid cells of seven sublines derived from individual colonies which arose in cocultures of male and female cells revealed that the mast cells and erythroid cells were both of the same sex in each of the seven sublines; this demonstrates the single cell origin of each colony and of the two lineages derived from it. We conclude that these nonadherent, factor-dependent cell lines are maintained by self-renewal and differentiation of bipotential progenitor cells apparently restricted to the erythroid and mast cell lineages.

Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase.

A method has been developed to quantitate megakaryocytopoiesis in culture by measuring acetylcholinesterase synthesized in vitro. Murine marrow cells, treated with diisopropylfluorosphosphate (DFP) to inactivate initial acetylcholinesterase (AchE) present in megakaryocytes and contaminating blood, were set up in Iscove's medium supplemented with 15% DFP-treated horse serum +/- pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) in 96-well microplates. Following the culture period, Triton X-100, dithiobisnitrobenzoic acid (DTNB), and acetylthiocholine iodide were added to each well. AchE synthesized in culture cleaved acetylthiocholine to thiocholine, which stochiometrically reduced the colorless indicator DTNB to a highly colored product. Thirty minutes following the addition of substrate, the plates were assayed for activity with a vertical recording photometer. When platelets, freshly prepared bone marrow cells, or cultured marrow were assayed by this method, a linear relationship was observed between optical density (OD) and the number of cells assayed. Moreover, a linear relationship between the number of AchE-positive megakaryocytes determined histochemically and AchE activity determined spectrophotometrically was observed. Red cells exhibited no activity. Inhibitor studies demonstrated that the activity measured was true AchE. Separation of marrow by density gradient centrifugation showed that the megakaryocyte enriched fraction contained all the AchE while the megakaryocyte depleted fraction contained none. From the data we conclude that this rapid, semiautomated method quantitates megakaryocytic AchE synthesis in culture, and that this method will be a useful assay system for the detection of factors that influence megakaryocytopoiesis.

Fluorescence Cytophotometric Analysis of Megakaryocytic Ploidy in Culture: Studies of Normal and Thrombocytopenic Mice

AbstractA system for the accurate and rapid measurement of the ploidy of cultured megakaryocytes derived from megakaryocytic colony-forming cells (CFU-M) has been developed. Thirty thousand murine marrow cells per milliliter were cultured for varying time periods in agar in the presence of horse serum and pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). To ensure the inclusion of all the megakaryocytic cells in the analysis, entire agar discs were transferred onto glass slides and dried. Cells of the megakaryocytic lineage were identified by staining for acetylcholinesterase (AchE) for two hours. Subsequently, the nuclei of the cells were stained using 1.7 × 10–5 mol/L chromomycin A3, a specific DNA-binding fluorochrome. Megakaryocytic colonies (≥ 2 AchE+ cells) were located under transmission light. The fluorescence emission of each cell of the colony was then measured by a photometer interfaced with a computer. The mean fluorescence emission of about 20 random granulocytes per slide was used as a 2N standard. There was no significant cell loss, quenching of fluorescence by AchE staining, or overlapping of colonies or cells. Approximately 100 megakaryocytes per hour could be analyzed. Modal ploidy of cultured megakaryocytes increased from 2N to 32N between days 3 and 6 in culture. Varying concentrations of PWM-SCM from 5% to 20% did not affect the ploidy distribution when examined at day 5. The heterogeneity of the ploidy of cells within colonies increased continuously with increasing cell numbers per colony. Clonal analyses of mean ploidy and ploidy heterogeneity did not show distinct types or classes of colonies; rather, the data show that megakaryocytic colonies are structured as a continuum. An inverse correlation was found between the number of cells constituting the colonies and their mean DNA content. To determine if shortterm in vivo exposure of CFU-M to a thrombocytopenic environment could affect the ploidy of their progeny, mice were given rabbit antimouse-platelet serum while control animals were given normal rabbit serum. Twenty-four hours after injection, marrow derived from these animals was cultured. At day 5, the ploidy distributions and ploidy heterogeneity were identical in both treated and control groups. Thus, factor(s) that promote CFU-M proliferation do not affect megakaryocytic endoreduplication, while stimuli that acutely influence megakaryocytic ploidy in vivo do not determine the ultimate ploidy potential of megakaryocytes derived from a CFU-M.

Factors affecting the generation of mast cells from multipotential colony-forming cells.

The cells responsible for the long-term in vitro generation of murine mast cells have been examined. Sequential analysis of all colony types obtained from cultures of spleen or bone marrow cells showed that only colonies derived from multipotential cells (mixed-erythroid colonies) or mast cell progenitors, contained cells responsible for mast cell generation in liquid cultures. Primary colony growth and subsequent maintenance of mast cells in liquid cultures was dependent upon pokeweed mitogen-stimulated spleen cell-conditioned medium (SCM). Mixed-erythroid colonies from 14-day cultures of spleen cells had the greatest capacity for mast cell generation. Analysis by clone splitting and transfer to high (20%) and low (2.5%) concentrations of SCM showed that the concentration of SCM used in either the primary colony culture or subsequent liquid culture phase altered both the proliferative capacity of the mast cells generated and the frequency of mast cell progenitors within individual mixed-erythroid colonies. Thus, mixed-erythroid colonies stimulated with 2.5% SCM contained the highest proportion of mast cell progenitors (34% of colonies) and when stimulated with 20% SCM, approximately fourfold higher numbers of mast cells were produced at weekly intervals from liquid cultures maintained in 2.5% SCM compared to parallel liquid cultures containing 20% SCM. These studies confirm the hemopoietic origin of mast cells and demonstrate that a factor(s) in SCM is able to modulate their proliferative potential.

Long-term culture of murine erythropoietic progenitor cells in the absence of an adherent layer.

Replication of multipotential stem cells in long-term murine bone marrow cell culture is known to depend on the development of an adherent stromal cell layer. In these conditions, restricted haematopoietic progenitor cells have also been generated for up to several months1-3. However, maturation is observed only in the granulocyte/macrophage and megakaryocyte lineages; erythropoiesis appears to be blocked at the earliest burst-forming unit (BFU-E) stage. Addition of exogenous erythropoietin (Epo) or anaemic mouse serum results in full erythropoietic maturation, but it is transient. We describe here a culture system in which production of erythropoietic progenitor cells can be maintained for over 6 months in the absence of an adherent stromal layer and in the absence of added Epo, but in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium (PWSCM). The data indicate that restricted erythroid progenitor cells exist which are capable of extensive self-renewal.

Factors altering the ability of multipotential hemopoietic colony-forming cells to self-generate or form progenitor cells

Individual in vitro colonies grown from multipotential cells have been analysed for their content of colony-forming cells including BFU-E, Mix-CFC and non-erythroid CFC. The CFC content of mixed colonies was higher in colonies grown from spleen cells than in colonies grown from bone marrow cells and was unaffected by the addition of thymocytes or postendotoxin serum. When the incubation period was extended from 7 to 14 days, CFC content of mixed colonies increased. However, plate mapping and reculturing of individual mixed colonies demonstrated that different subsets of mixed colonies were present in day 7 and day 14 cultures. Although the number of mixed colonies developing decreased when stimulated by decreasing concentrations of pokeweed mitogen-stimulated spleen cell conditioned medium (SCM) the mean number of cells per colony remained unaltered and the content of CFC in such colonies increased. With the SCM concentration range tested, as the SCM concentration was decreased, the absolute number of mixed colonies containing CFC rose from 0.6 per culture to 2.4 per culture. Over this same SCM concentration range the mean number of CFC per mixed colony increased from 1.3 to 185.2. These data suggest that amplification within mixed colonies of the number of cells capable of colony formation may occur at the expenses of the production of differentiating cells.

Clonal origin of murine hemopoietic colonies with apparent restriction to granuclocyte-macrophage-megakaryocyte (GMM) differentiation.

We characterized murine hemopoietic colonies consisting of granulocytes, macrophages, megakaryocytes, and blast cells and yet lacking erythroid elements. Mouse marrow or spleen cells were cultured in methylcellulose media in the presence of 10% (v/v) pokeweek mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) and 2 units/ml erythropoietin for 8 days. Granulocyte-macrophage-megakaryocyte (GMM) colonies could be distinguished from granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies because the former lacked the typical appearance of bursts with red color. Analysis of Y-chromosomes in mixing experiments with male and female marrow cells confirmed the clonal nature of the GMM colonies. Differential counts of GMM colonies revealed varying, but significant, numbers of blast cells in all of the day-8 and day-12 colonies and in seven out of ten day-14 GMM colonies. In general, the percentages of blast cells were inversely related to the length of incubation in culture. Replating experiments confirmed the absence of late erythroid precursors such as CFU-E and normoblasts in all of the 50 day-8 GMM colonies. However, six out of the 50 GMM colonies contained early progenitors capable of erythroid expression, such as BFU-E, CFU-EM, CFU-GEM, and CFU-GEMM. In contrast, the three day-14 GMM colonies which did not reveal blast cells failed to produce secondary colonies. Thus, while the progenitors for the latter colonies are restricted to only granulocyte-macrophage-megakaryocyte differentiation, some of the apparent GMM colonies containing blast cells may have originated in early progenitors close to pluripotent stem cells. Detailed cytological analyses and replating experiments are necessary for characterization of true differentiation potentials of mixed colonies in culture.