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Immature megakaryocytes in the mouse: physical characteristics, cell cycle status, and in vitro responsiveness to
Abstract:
The heterogeneity among immature megakaryocytes has been examined by physical properties, cell cycle status, and responsiveness to thrombopoietic stimulatory factor. Three types of immature megakaryocytes exist that can be recognized by acetylcholinesterase staining, nuclear shape, high nucleus/cytoplasm ratio, and small size (8--18 mu) with respect to mature megakaryocytes (greater than 18 mu). These three acetylcholinesterase-containing cell types are distinguished by their nuclear configuration: a round, indented, and lobed nucleus. The lobed cell type was found to overlap with and enhance detection of megakaryoblasts (stage I megakaryocytes). These cells had a sedimentation velocity range of 3.5--19.0 mm hr-1 and a density range of 1.072--1.095 g cm-3. Separation of these three classes of immature megakaryocytes was achieved by equilibrium density centrifugation with modal buoyant densities of 1.079 g cm-3 (round), 1.084 g cm-3 (indented), and 1.089 g cm-3 (lobed). In the presence of thrombopoietic stimulatory factor, the round nucleated cells, but not the indented or lobed nuclei morphology, were observed to develop into large mature megakaryocytes in 60-hr semisolid cell cultures. Development of two cell groups, or colonies of megakaryocytes, was not observed during this in vitro incubation period. In vivo treatment with hydroxyurea indicated that 57.5% +/- 19% of the round nucleus form were actively synthesizing DNA. No reduction in the numbers of indented or lobed nucleus forms were observed following hydroxyurea treatment. The data in this report strongly support the concept that these three types of immature megakaryocytes reflect the early maturation stages occurring in megakaryocyte differentiation.
Insights
Three distinct immature megakaryocyte types, identified by nuclear shape, represent early differentiation stages. Only round-nucleated cells mature into functional megakaryocytes, indicating a specific developmental pathway.
Area of Science:
- Hematology
- Cell Biology
- Developmental Biology
Background:
- Immature megakaryocytes exhibit heterogeneity.
- Distinguishing these early cell stages is crucial for understanding megakaryopoiesis.
Purpose of the Study:
- To characterize the heterogeneity of immature megakaryocytes.
- To investigate the differentiation potential and cell cycle status of distinct immature megakaryocyte populations.
Main Methods:
- Analysis of physical properties (size, density, sedimentation velocity).
- Acetylcholinesterase staining and nuclear morphology assessment.
- In vitro culture with thrombopoietic stimulatory factor and in vivo hydroxyurea treatment.
Main Results:
- Three immature megakaryocyte types identified by nuclear shape (round, indented, lobed) and acetylcholinesterase staining.
- Equilibrium density centrifugation separated these types with distinct modal buoyant densities.
- Only round-nucleated cells differentiated into mature megakaryocytes in vitro.
- Round-nucleated cells showed active DNA synthesis post-hydroxyurea treatment, unlike other types.
Conclusions:
- The three identified immature megakaryocyte populations represent sequential stages in megakaryocyte differentiation.
- The round-nucleated cell type is a proliferative precursor that gives rise to mature megakaryocytes.