Small Molecule Supplements Improve Cultured Megakaryocyte Polyploidization by Modulating Multiple Cell Cycle Regulators

Abstract

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.

Figure 1

Effect of small molecules on MK polyploidization in megakaryocytic cells. (a) Morphological difference of the cells after treatment with small molecules was shown by Wright–Giemsa staining. Cell diameter of over 50 cells from five random views was measured (scale bars: 20 μm). (b) DNA ploidy analysis by flow cytometry. Ploidy (≥8N) was compared with control in three cell lines. (c) The proliferation of K562 and MEG-01 cells with different small molecules evaluated by CCK8 assay. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 2

Effect of small molecules on MK polyploidization in human MK progenitors (MK-PROs). (a) MK-PROs DNA ploidy analysis (4N and ≥8N) after different small molecules treatment. Representative flow cytometry plots are shown in the upper panel. Statistical analysis was made by comparing with the control group. (b) Morphology and cell diameter of MK-PROs after small molecules treatment for 10 days (scale bars: 50 μm). (c) The proportion of CD61⁺ cells after treated with four small molecules was normalized to control. (d) The apoptosis analysis by flow cytometry in MK-PROs. Histogram of the proportion of early apoptosis and late apoptosis in MK-PROs treated with or without small molecules is showed in the lower panel. (e) The cell proliferation with or without small molecules. Results are means and SEM from biological replicates (n = 8). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 3

The combination of small molecules for polyploidization in MKs. (a) Ploidy analysis by flow cytometry. Ploidy (≥8N) after the combination of small molecule treatment was compared with control in three megakaryocytic cells. Representative flow cytometry plots of RRI + SI treatment or control are shown in the lower panel. (b) Morphology of stained polyploid megakaryocytes in UT-7 cells treated with RRI and SI (scale bars: 20 μm). (c) Cell diameter of the MK-PROs after small molecule combinations treatment for 10 days. (d) The proportion of CD61⁺ cells from MK-PROs treated with the combined small molecules, normalized to control. (e) The early apoptosis and late apoptosis were detected by flow cytometry. Lower panel: the proportion of apoptosis in MK-PROs treated with combined small molecules, compared with control. (f) Ploidy (4N and ≥8N) in MK-PROs treated with RRI and SI was compared with control. Results are means and SEM from biological replicates (n = 7). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 4

Expression of cell cycle-related proteins and cytoskeletal proteins after RRI and SI treatment. (a) Expression of cyclin B1, cyclin D1 cyclin E1, β-actin, p21, and p53 detected by Q-PCR in MEG-01 cells and MK-PROs. (b) The p53, p21, β-actin, and cyclin B1 expression detected by western blot in MEG-01, UT-7, and MK-PROs. The intensity of each band was digitalized and compared by Image Lab software. (c) Schematic diagram of the relationship of small molecules and cell cycle-related proteins. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.