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Analysis of murine megakaryocyte colony size and ploidy: effects of interleukin-3
G M Segal1, T Stueve, J W Adamson
1Department of Medicine, Oregon Health Sciences University, Portland 97201.
Abstract:
Megakaryocytes develop from diploid precursor cells that, after variable numbers of mitoses, cease cell division and then undergo synchronous nuclear endoreduplication (endomitosis). Megakaryocyte colony formation represents an in-vitro model of these processes in which the number and ploidy of colony cells reflect the activity of the mitotic and endomitotic pathways, respectively. We have analyzed the size and ploidization of murine megakaryocyte colonies grown in agar and examined the influence of interleukin-3 (IL-3) on these parameters. Colonies were identified in situ by staining for acetylcholinesterase and the ploidy of colony cells was determined by fluorescence cytophotometry. More than 98% of the megakaryocytes that developed in culture could be analyzed. In cultures of unfractionated marrow cells stimulated by either pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM, a crude source of megakaryocyte colony-stimulating activity) or IL-3, the modal ploidy of day-5 colony megakaryocytes was 16N (range 2N-128N). The distribution of colony size was described by an inverse exponential relationship. Colony size and geometric mean ploidy were inversely correlated under conditions of maximal stimulation with PWM-SCM and at all concentrations of IL-3 tested. Increasing concentrations of IL-3 in cultures of either unfractionated marrow cells or nonadherent T-lymphocyte-depleted (NATD) marrow cells stimulated similar dose-dependent increases in the mean size and numbers of megakaryocyte colonies but did not significantly alter their ploidy distribution. However, the mean ploidy of colony megakaryocytes in IL-3-stimulated cultures of NATD marrow cells was significantly less (P less than 0.001) than the mean ploidy of megakaryocytes in IL-3-stimulated cultures of unfractionated marrow cells. The mean ploidy of megakaryocytes, which developed in PWM-SCM-stimulated cultures, was not affected by initial accessory cell depletion. We conclude that the size and ploidy characteristics of day-5 murine megakaryocyte colonies are structured as continua and that IL-3 stimulates an increase in mean colony size and numbers without affecting ploidization. T-lymphocytes and adherent cells elaborate an activity which promotes endomitosis in vitro; factors in PWM-SCM can substitute for this activity.
Insights
Interleukin-3 (IL-3) increases megakaryocyte colony size and number but not ploidy. Accessory cells promote endomitosis, which can be substituted by factors in pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM).
Area of Science:
- Hematopoiesis
- Cell Biology
- Molecular Biology
Background:
- Megakaryocytes originate from diploid precursors undergoing mitosis and endoreduplication.
- Megakaryocyte colony formation in vitro models these developmental processes.
- Colony cell number and ploidy indicate mitotic and endomitotic pathway activity.
Purpose of the Study:
- To analyze size and ploidy of murine megakaryocyte colonies.
- To investigate the influence of interleukin-3 (IL-3) on megakaryocyte colony parameters.
- To understand the role of accessory cells in megakaryocyte endomitosis.
Main Methods:
- Murine megakaryocyte colonies were cultured in agar.
- Colonies were identified by acetylcholinesterase staining.
- Cell ploidy was determined using fluorescence cytophotometry.
Main Results:
- IL-3 increased megakaryocyte colony size and number dose-dependently without altering ploidy distribution.
- Colony size and mean ploidy were inversely correlated under maximal stimulation and with IL-3.
- Accessory cells (T-lymphocytes and adherent cells) promote endomitosis in vitro.
Conclusions:
- Megakaryocyte colony size and ploidy exist as continua.
- IL-3 primarily influences megakaryocyte proliferation, not endoreduplication.
- Accessory cell-derived factors enhance endomitosis, with PWM-SCM factors offering a substitute.