FCS files for S3 Fig. C, D.

Millions of platelet units are needed each year to manage thrombocytopenia and other conditions linked to excessive bleeding. These life-saving treatments still depend entirely on donated platelets, despite the numerous shortcomings associated with them, such as limited shelf life, supply shortages, unpredictable functionality, potential for infection, as well as immune-incompatibility issues. These challenges could be overcome with universal donor platelets generated from human induced pluripotent stem cell (hiPSC)-derived megakaryocytes (MKs). We recently developed expandable hiPSC-derived megakaryocytic cell lines (imMKCLs) as a potentially unlimited source for platelet production. imMKCL-derived platelets are functional and have already been tested in patients. In this study, we demonstrate through single-cell time-course imaging that imMKCL maturation is heterogeneous and asynchronous, with only a few imMKCLs generating platelets at any given time under static culture conditions. Using a chemical screen, we identify microtubule (MT) destabilizing agents, including vincristine (VCR), as promising hits, with a larger proportion of VCR-exposed imMKCLs developing proplatelet extensions and more platelets being produced per imMKCL. VCR use reduces the MT content of imMKCLs and results in the production of platelets with a diminished peripheral MT ring structure. Nevertheless, these platelets are functional, as evidenced by their normal response to agonists, their ability to attach to and spread on fibrinogen-coated surfaces, and their capacity to restore hemostasis in vivo. Interestingly, we also observed a negative correlation between the MT content of imMKCLs and platelet yields when we compared imMKCLs differentiated under static conditions (MThigh, low yield) to our turbulence-optimized VerMES™ bioreactor (MTlow, high yield). Taken together, our findings highlight the importance of MT dynamics in megakaryocyte biology, provide a possible explanation for the still poorly understood link between vinca alkaloid in vivo use and thrombocytosis, and bring us closer to realizing the clinical potential of affordable, off-the-shelf hiPSC-derived platelets.

每年都有数以百万计的血小板单位被用于治疗血小板减少症（thrombocytopenia）以及其他与过度出血相关的病症。这类挽救生命的治疗手段目前仍完全依赖捐赠血小板，但该方式存在诸多弊端：如储存有效期有限、供应短缺、功能难以预测、存在感染风险以及免疫不相容等问题。通过人类诱导多能干细胞（human induced pluripotent stem cell, hiPSC）衍生的巨核细胞（megakaryocytes, MKs）制备通用供体血小板，有望克服上述挑战。

我们团队近期开发了可扩增的hiPSC衍生巨核细胞系（imMKCLs），作为血小板生产的潜在无限来源。由imMKCLs衍生的血小板具备生理功能，且已在患者中完成测试。本研究通过单细胞时序成像（single-cell time-course imaging）技术证实：在静态培养条件下，imMKCLs的成熟过程呈现异质性与不同步性，任意时刻仅少数imMKCLs能够生成血小板。

本研究通过化学筛选鉴定出包括长春新碱（vincristine, VCR）在内的微管（microtubule, MT）解聚剂为潜在有效靶点；经VCR处理的imMKCLs中，具备前血小板延伸结构的细胞比例更高，且每个imMKCL生成的血小板数量更多。VCR处理会降低imMKCLs的微管含量，并使得生成的血小板外周微管环结构出现缺损。尽管如此，这些血小板仍具备正常生理功能，具体表现为对激动剂的应答正常、可在纤维蛋白原（fibrinogen）包被的表面黏附并铺展，且能够在体内恢复止血（hemostasis）功能。

值得注意的是，本研究将静态培养条件下分化的imMKCLs（高微管含量，低血小板产率，MThigh）与湍流优化的VerMES™生物反应器中培养的imMKCLs（低微管含量，高血小板产率，MTlow）进行对比后发现，imMKCLs的微管含量与血小板产率呈负相关。

综合来看，本研究的结果阐明了微管动力学在巨核细胞生物学中的重要作用，为长期以来尚未明确的长春花生物碱（vinca alkaloid）体内使用与血小板增多症（thrombocytosis）之间的关联提供了潜在解释，并进一步推动了可负担、可现货供应的hiPSC衍生血小板的临床转化进程。