REPLICATION AND ENDOREPLICATION IN DEVELOPING MEGAKARYOCYTES IN-VITRO

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 mononucle ar cells highly enriched in hematopoietic progenitors were cultured in liquid cultures and plasma clots in the presence of either interleuki n-3 (IL-3) and stem cell factor (SCF) or medium conditioned by blood m ononuclear cells stimulated by phytohemagglutinin (PHA). In plasma dot s, megakaryocytic (MK) colonies appeared first on day 5 and reached a maximum by day 8, whereas the number of cells per colony increased unt il day 10, indicating that there was a single wave of MK colony format ion. In liquid cultures, the first immunologically recognizable megaka ryocytes appeared on day 5 and expressed GPIIb/lIIa 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 repl icating. Their replicative potential decreased with age, however, so t hat between days 6 and 11, a linear correlation was noted between the logarithm of the percentage of megakaryocytes with replicative capacit y and their age in culture. Replication ceased completely after day 10 . In the presence of IL-3, polyploid megakaryocytes appeared at the sa me time that GPIIb/IIIa was expressed, and the megakaryocyte distribut ion into ploidy classes remained unchanged until day 20. In the presen ce of PHA-leukocyte conditioned medium (PHA-LCM), ploidy of megakaryoc ytes was shifted toward higher classes after day 6, and the process of endoreplication was completed by day 10. No changes in ploidy distrib ution were noted between days 10 and 20. These findings indicate that in the cohort of megakaryocytes derived from colony-forming units-mega karyocyte (CFU-MK), endoreplication can occur at an early stage of dev elopment, proceeds synchronously with replication, and is completed be fore the megakaryocytes exhaust their replicative potential.