Optimizing Megakaryocyte Polyploidization in Culture by Targeting Multiple Pathways of Cytokinesis
Authors: Avanzi Mauro P. , Chen Amanda , Haugh Alexandra , Mitchell W. Beau 
Published: October 2019

Abstract
Abstract 4820

Introduction:
The large-scale laboratory production of platelets for transfusion purposes using cord blood stem cells is one of goals of stem cell research. One step toward this goal will be to produce and expand megakaryocytes with high ploidy which are capable of releasing higher amounts of platelets than low ploidy cells. Megakaryocyte polyploidization requires a combination of distinct cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. Normal bone marrow megakaryocytes reach a DNA content of 128N, with a modal ploidy of 16N. In contrast, cultured human megakaryocytes have typically reached a maximum ploidy of 16N with the majority of cells at 2N/4N. In this study we combined the inhibition of each of the principle mechanisms driving cytokinesis with the goal of driving polyploidization of cultured CD34+ umbilical cord blood cells. The effects of inhibiting each cytokinesis process was characterized by its effects on cell expansion and ploidy, and the additive or inhibitory effects of combining inhibition of multiple processes was determined. The results of this study provide a strategy to produce high ploidy megakaryocytes and provide new insight into the mechanisms underlying megakaryocyte polyploidization.

Methods:
Human cord blood derived megakaryocytes were cultured with thrombopoietin (TPO) and stem cell factor (SCF) along with reagents that inhibit different mechanisms of the cytokinesis process: Rho-Rock inhibitor, Y27632 (RRI); Src-inhibitor, SU6656 (SI); Nicotinamide (NIC); Aurora-B inhibitor, ZM447439 (ABI); and Myosin Light Chain Kinase Inhibitor (MLCKI). Reagents were added in one single day (day 8) or on three consecutive days (days 8–10) and ploidy was analyzed on day 11 of culture with PI/RNase. Combinations of reagents were used in order to determine their additive or inhibitory interactions and to maximize megakaryocyte ploidy.

Results:
Treatment with RRI resulted in the highest megakaryocyte ploidy. Up to 48% of megakaryocytes were high ploidy (≥ 8N), with highest ploidy ≥ 64N (p = 0.0007 vs. control). Treatment with NIC resulted in up to 24% of cells reaching high ploidy, with a maximum ploidy of ≥ 32N (p = 0.003 vs. control). Treatment with SI resulted in up to 24% megakaryocytes reaching high ploidy, with a maximum ploidy of ≥ 32N (p = 0.026 vs. control). Treatment with ABI resulted in up to 28% of megakaryocytes reaching high ploidy, with a maximum ploidy of ≥ 32N (p = 0.018 vs. control). The MLCKI had no effect in final ploidy. The combinations of reagents all significantly increased both percentage of cells reaching high ploidy and the highest ploidy compared to control. However, none of the combinations achieved a more robust effect in final ploidy than the RRI alone. However, the combination of MLCKI with SI, NIC and ABI increased the high ploidy cells up to 51.3% with a maximum ploidy ≥ 64N (p < 0.0001 vs. control) (Table 1).

Conclusion:
The RRI proved to be the most effective agent in driving umbilical cord-derived megakaryocyte polyploidization. All other reagents tested, except MLCKI, also moderately increased megakaryocyte ploidy. RRI and MLCKI, both reagents that inhibit the late stages of cytokinesis and furrow formation, resulted in opposite ploidy outcomes, suggesting that RRI acts on ploidy through pathways other than inhibition of myosin activation. While MLCK is active primarily in the late stages of cytokinesis, the Rho/Rock pathway overlaps with other signaling pathways involved in cytokinesis, including src activation in actin polymerization and Aurora kinase activity in microtubule formation. Although inhibition of myosin light chain activation by itself did not appear to drive polyploidization, the combination of MLCKI with other cytokinesis inhibitors increased polyploidization to the same extent as RRI. Our results indicate that induction of high ploidy in cord blood derived megakaryocytes involves a combination of distinct cytokinesis pathways.

Disclosures:
No relevant conflicts of interest to declare.