Kinetic analysis of megakaryocyte numbers and ploidy levels in developing colonies from mouse bone marrow cells

. The kinetics of megakaryocyte formation from mouse bone marrow cells in semi‐solid medium was studied directly in the culture dish by staining the cells for acetylcholinesterase after drying the cultures. A WEHI‐3 cell‐conditioned medium (WEHI‐3 CM) was used as a general source of stimulus for megakaryocyte colony formation. The addition of peritoneal exudate supernatant as well as WEHI‐3 CM increased the frequency of megakaryocyte colonies detected. Colonies containing acetylcholinesterase‐positive cells were first detected at day 3. Maximum numbers of 25–40 megakaryocyte colonies per 10 5 nucleaet mouse bone marrow cells were observed from days 7 to 11. The mean number of cells within each colony increased progressively with time of culture, and a modal range of 11–20 cells was obtained by day 7. Between 3 and 200 cells per colony were generally detected. A continuous distribution of the number of megakaryocytes per colony suggests that the clonable precursor cells are not synchronized either with respect to maturation stage or with respect to their capability to undergo nuclear endoreduplication. The addition of peritoneal exudate supernatant to the cell cultures increased the DNA levels of megakaryocytes grown in the presence of WEHI‐3 CM but did not affect the number of cells per colony. The DNA content of colony megakaryocytes was measured after staining the cells with Feulgen reagent. A modal DNA value of 8 N was observed between days 4 and 7 for megakaryocytes stimulated with WEHI‐3 CM. In the presence of both WEHI‐3 CM and peritoneal exudate supernatant, the DNA content of megakaryocytes increased with the time of cell culture. Modal DNA values increased from 8 N at days 4 and 5, to 16 N by day 6. In these optimally stimulated cultures, 44% of colony megakaryocytes were 32 N or greater, a proportion higher than in normal bone marrow, but similar to that seen in the marrow of acutely thrombocytopenic animals. It is concluded that megakaryocytopoiesis in cell cultures is not a strictly controlled process with respect to cell division and endomitosis and that when certain culture conditions are employed, megakaryocyte development in vitro might reflect that seen in a stressed animal condition.