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Replication and endoreplication in developing megakaryocytes in vitro
P Angchaisuksiri1, P L Carlson, E B Day
1Department of Medicine, H. H. McGuire Department of Veterans Affairs Medical Center, Richmond, VA 23249.
Abstract:
To study the relation in time between replication and endoreplication and the relation between appearance of platelet-specific proteins and endoreplication in maturing megakaryocytes, peripheral blood mononuclear cells highly enriched in hematopoietic progenitors were cultured in liquid cultures and plasma clots in the presence of either interleukin-3 (IL-3) and stem cell factor (SCF) or medium conditioned by blood mononuclear cells stimulated by phytohemagglutinin (PHA). In plasma clots, megakaryocytic (MK) colonies appeared first on day 5 and reached a maximum by day 8, whereas the number of cells per colony increased until day 10, indicating that there was a single wave of MK colony formation. In liquid cultures, the first immunologically recognizable megakaryocytes appeared on day 5 and expressed GPIIb/IIIa and thrombospondin only, but all other platelet-specific protein markers appeared within 24 hours. Replating cells from liquid medium into plasma clots showed that 92 +/- 8% of day 6 GPIIb/IIIa-positive cells are capable of replicating. Their replicative potential decreased with age, however, so that between days 6 and 11, a linear correlation was noted between the logarithm of the percentage of megakaryocytes with replicative capacity and their age in culture. Replication ceased completely after day 10. In the presence of IL-3, polyploid megakaryocytes appeared at the same time that GPIIb/IIIa was expressed, and the megakaryocyte distribution into ploidy classes remained unchanged until day 20. In the presence of PHA-leukocyte conditioned medium (PHA-LCM), ploidy of megakaryocytes was shifted toward higher classes after day 6, and the process of endoreplication was completed by day 10. No changes in ploidy distribution were noted between days 10 and 20. These findings indicate that in the cohort of megakaryocytes derived from colony-forming units-megakaryocyte (CFU-MK), endoreplication can occur at an early stage of development, proceeds synchronously with replication, and is completed before the megakaryocytes exhaust their replicative potential.
Insights
Megakaryocyte (MK) development involves replication and endoreplication occurring together. Platelet protein expression and endoreplication in MKs are closely linked, completing before replicative potential is exhausted.
Area of Science:
- Hematopoiesis
- Cell Biology
- Molecular Biology
Background:
- Megakaryocytes (MKs) are essential for platelet production.
- Understanding MK maturation, including replication and endoreplication, is crucial for hematopoiesis research.
Purpose of the Study:
- To investigate the temporal relationship between replication and endoreplication in maturing megakaryocytes.
- To examine the link between platelet-specific protein appearance and endoreplication during MK development.
Main Methods:
- Culturing peripheral blood mononuclear cells enriched in hematopoietic progenitors in liquid cultures and plasma clots.
- Utilizing growth factors like IL-3 and SCF, or PHA-leukocyte conditioned medium (PHA-LCM).
- Assessing megakaryocyte colony formation, cell proliferation, replication capacity, and ploidy distribution over time.
Main Results:
- Megakaryocytic colonies formed early, with cell numbers increasing later, indicating a single wave of colony formation.
- Platelet-specific proteins appeared rapidly after initial MK recognition.
- Megakaryocytes retained replicative capacity for several days, with potential decreasing over time.
- Endoreplication occurred early, synchronously with replication, and was completed before replication ceased.
- IL-3 supported stable ploidy, while PHA-LCM induced a shift towards higher ploidy classes.
Conclusions:
- Endoreplication in megakaryocytes begins early in development and proceeds concurrently with replication.
- The process of endoreplication is completed before megakaryocytes exhaust their replicative capacity.
- These findings provide insights into the coordinated processes governing megakaryocyte maturation and platelet formation.