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S. Bhatlekar et al.
ly 10% to approximately 19% (Figure 6C), suggesting BCL2L2 enables mature platelet production.
Platelet-like particles were stimulated with thrombin to assess their ability to activate αIIbβ3 and release α-gran- ules. PLP from both control and BCL2L2 lentiviral con- structs demonstrated cell activation above background (Figure 5D and E, and flow cytometry plots in Online Supplementary Figure S6); the signal was small but consis- tent in PLP with control lentivirus. Importantly, among these CD41a+ and CD42a+ PLP, MK BCL2L2 overexpres- sion enhanced thrombin-induced activation compared to control lentivirus (Figure 6D and E, and flow cytometry plots in Online Supplementary Figure S6). PAC1 binding and P-selectin expression in PLP treated with buffer instead of thrombin were not altered by BCL2L2 overexpression (data not shown).
Discussion
There has been exciting progress in the generation of MK and platelets via the use of induced pluripotent stem cells, immortalized MK cell lines, and bioreactors,1,23,24 but cultured human CB MK remain an important tool for gain- ing a deeper understanding of MKpoiesis and platelet pro- duction. Apoptosis of MK in a culture system remains a major obstacle to progress in the in vitro generation of MK and platelets.6 A great deal of our understanding about the apoptosis genes regulating MKpoiesis and PB platelet
number is derived from work in murine systems,12,20,25-27 while relatively less is known about in vitro cultured MK apoptosis. The major finding in the current study is that the pro-survival gene BCL2L2 restrains apoptosis in cul- tured human MK, regulates PPF, and is associated with platelet number in healthy humans. We also found that suspensions of CB-derived MK are a useful system for assessing candidate gene function by flow cytometric analysis, and that BCL2L2 overexpression induced increases in agonist-induced signaling responses in PLP. BCL2L2 thus becomes another potential target for enhanc- ing MK yields in vitro, which may benefit both basic research on MKpoiesis and the long-term goal of produc- ing platelets for transfusion into patients.
Two distinct populations of cells invariably emerged during our CB MK cultures that were easily distinguished by flow cytometry logarithmic scale forward and side scatter measures. Electron microscopic analysis of the LLG MK (Figure 2) demonstrated they were most similar to pri- mary BM MK, and resembled those observed by Cramer et al.28 and Chantelot-Bellanne et al.29 In contrast, the SHG population represented primarily apoptotic cells resem- bling the senescent MK observed by Radley et al., where the nucleus fragmented but the cells appeared to change shape and 'round up' rather than fragment.30 Membrane degradation during apoptosis likely contributed to the loss of MK markers (Figure 1B). The most important functional differences between LLG and SHG were the ability to form proplatelets and 'signal' in response to platelet ago-
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Figure 5. BCL2L2 overexpression reduces megakaryocyte (MK) apoptosis. (A-F) Transduction of MK cultures with empty lentiviral vector (Ctrl lenti) or lentiviral vector containing BCL2L2. (A) Overexpression of BCL2L2 mRNA was confirmed by quantitative polymerase chain reaction (qPCR). Fold change of BCL2L2 mRNA levels was determined compared to Ctrl lenti (n=5). (B) Representative western blot image for Bcl-w protein (encoded by BCL2L2 gene) for BCL2L2 overexpression compared to Ctrl. (C) Densitometric analysis of western blots for BCL2L2 overexpression compared to Ctrl (n=5). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalizer for densitometric analysis. (D) The percentage of annexin V-positive (+) CD41a+ MK was determined for BCL2L2 over-expressed and Ctrl cells by flow cytometry (n=5). (E) Representative image of proplatelet formation in Ctrl and BCL2L2 over-expressed cells. Image shows α-tubulin staining (red) and nuclear stain, DAPI (blue). White arrows show proplatelet-forming MK. (F) Quantification of proplatelet forming (PPF) MK amongst total cells blinded to treatment group (n=5).
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haematologica | 2019; 104(10)