In vitro regulatory mechanisms for cytoplasmic maturation of murine megakaryocytes derived from colony-forming units megakaryocyte (CFU-M).

We studied the effects of recombinant erythropoietin (r-Epo) and thrombocytopenic serum (TS) on the cytoplasmic maturation of megakaryocytes derived from colony-forming units megakaryocyte (CFU-M). Serotonin content, ATP content, acetylcholinesterase (Ach-E) activity per megakaryocyte, and electron microscopic analysis were selected as markers of cytoplasmic maturation. Megakaryocytes induced by pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) alone showed low levels of ATP content and Ach-E activity, which did not increase during culture, as well as a low level of serotonin content, which gradually accumulated during day 7 of culture. When r-EPo was added to the culture system on day 3 after megakaryocytic colony formation with PWM-SCM, the serotonin content in megakaryocytes increased markedly but ATP content and Ach-E activity did not increase significantly. In contrast, TS caused an increase in ATP content and Ach-E activity, but did not cause an increase in serotonin content. Electron microscopic analysis showed that the demarcation membrane system (DMS) developed defectively only in local areas with the addition of r-Epo and PWM-SCM, whereas the DMS developed normally and dense granules were generated to near normal with the addition of TS. Recombinant Epo may act on the early stage of cytoplasmic maturation, whereas TS may act on the late stage of cytoplasmic maturation. The results shown herein suggest that at least two different factors may be necessary for full in vitro cytoplasmic maturation of megakaryocytes derived from CFU-M.