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S. Bhatlekar et al.
Supplementary Figure S4), they do not seem to be 'exhaust- ed' MK post release of proplatelets.
Sim et al. used adult mobilized HSPC to culture MK that also gave rise to populations of functional and apoptotic MK.17 Our work differs from this primarily by studying the molecular mechanism of apoptosis. We are unaware of direct comparisons among any source of MK (neonatal, adult, murine, embryonic stem cells, etc.) in terms of the degree of apoptosis in culture, but it is reasonable to con- sider that some molecular mechanisms will be shared. In this regard, our data show no statistically significant dif- ference between BCL2L2 levels in neonatal and adult MK (P>0.47), and adult MK also display more PPF with BCL2L2 overexpression (Online Supplementary Figure S8). As with MK, there is no statistically significant difference between BCL2L2 levels in neonatal and adult platelets.39
The finding of a significant positive correlation between human platelet BCL2L2 levels and PB platelet counts (Figure 5A) supports a mechanism whereby Bcl-w increas- es MK PPF which causes increased blood platelets. Numerous apoptosis genes have been studied for effects on Mkpoiesis.8-13,19,40,41 We observed that pro-apoptotic genes BAX and BAK1 showed little to no change in expres- sion from day 6 to day 13 in MK cultures (Online Supplementary Figure S9A and B). However, similar to BCL2L2, anti-apoptotic gene BCL2L1 showed increased expression in LLG over time, and was higher at day 13 in LLG compared to SHG (Online Supplementary Figure S9C). Taken together, these studies suggest BCL2L1 and BCL2L2 restrain apoptosis and that this may be the key mecha- nism for prolonging MK survival in the late stages of dif- ferentiation in culture. We cannot exclude a role for other apoptosis genes in Mkpoiesis and platelet production. It is also reasonable to consider that, like Bcl-xL and Bak, Bcl- w may also impact the life span of the human PB platelet.20 However, additional studies are needed to address this possibility.
The BCL2L2-induced increase in MK PPF led to experi- ments assessing production of PLP. BCL2L2 overexpres- sion roughly doubled the number of CD41a+ PLP and increased the ability of these PLP to show a functional response when activated with thrombin (Figure 5C-E). However, we did not observe any significant correlation between in vivo BCL2L2 expression with other platelet
agonists such as ADP, PAR1-AP and PAR4-AP (www. Plateletomics.com, as reported by Simon et al.40). Nevertheless, we would emphasize that the percentages of functional PLP were small, although consistent and sta- tistically significant.
Our findings have several research and clinical implica- tions. Firstly, we have shown that simple gating on flow cytometric forward and side scatter measurements allows the identification of the subset of viable and functional MK. This approach represents a substantial improvement in the signal-to-noise read-outs to test the functionality of candidate MK/platelet genes identified in GWAS. Secondly, promotion of apoptosis by inhibiting of Bcl-2 family members is under investigation for the treatment of a variety of malignancies;41-43 a limiting toxicity has been thrombocytopenia.42,43 Bcl-2 family members, MCL1 and BCL2L1 are the most frequently amplified in 26 tumor types,44,45 whereas BCL2L2 amplification is rare.46 Perhaps selective targeting of Bcl-2 family members that spares Bcl-w could minimize thrombocytopenia. Lastly, progress toward the manufacture of in vitro platelets may benefit from the pro-survival benefits on MK generation and PPF of Bcl-w. BCL2L2 overexpression may have synergistic or additive effects when used in combination with an antag- onist of the aryl hydrocarbon receptor (StemRegenin 1), BCL2L1 overexpression, abscisic acid, and other factors that have been shown to increase numbers of proplatelet- forming MK in culture.1,47,48
Funding
This study was supported by a grant from the National Institutes of Health National Heart, Lung and Blood Institute (HL116713) and funding from the Cardeza Foundation of Hematologic Research. The authors thank Ms. Lin Ma, Thomas Jefferson University, and Ms. Sarah Hugo, University of Utah for their help isolating cord blood stem cells; Dr. Xianguo Kong, Thomas Jefferson University for designing lentiviral vectors; Ms. Diana Lim, Molecular Medicine Program from University of Utah, for drafting the figures and consultation on data display.
Acknowledgments
The authors also thank the University of Utah Flow Cytometry Facility in addition to the National Cancer Institute through Award Number 5P30CA042014-24.
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