(74d) Modulating Megakaryocyte Adhesion for Improved Proplatelet Formation

Megakaryocytes generate long, cytoplasmic extensions, known as proplatelets, to package platelets and release them into the circulation. In vivo, this process occurs in the bone marrow vascular niche. Mature megakaryocytes express receptors for many of the ligands present in this niche, suggesting that cell adhesion may play an important role in proplatelet formation. We aim to improve proplatelet formation for the purpose of in vitro platelet production. We found that the CHRF megakaryoblastic cell line and megakaryocytes derived from CD34+ mobilized peripheral blood stem and progenitor cells formed proplatelets on a tissue-culture treated surface, but not on a neutral, hydrophilic polymer that inhibits protein adsorption and cell adhesion. Importantly, CHRF cells cultured on the nonadhesive surface attained much higher mean ploidy by day 11 (8.6±0.5N vs. 5.8±0.6N, P<0.005) and maintained higher viability. Subsequent transfer to tissue-culture plastic yielded faster and more extensive proplatelet formation compared to cells initially cultured on tissue-culture plastic, with a maximal increase for day-5 transfer. Interestingly, acetylated alpha tubulin was abundant in the proplatelet extensions of CHRF cells on tissue-culture plastic, but was present at a minimal level in CHRF cells on the nonadhesive surface. We are currently exploring the role of tubulin acetylation, a hallmark of microtubule stability, in polyploidization and proplatelet formation.