Division of Hematology, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA.
Department of Pharmacology and.
Blood Adv. 2018 Mar 27;2(6):597-606. doi: 10.1182/bloodadvances.2017010975.
In vitro-grown megakaryocytes for generating platelets may have value in meeting the increasing demand for platelet transfusions. Remaining challenges have included the poor yield and quality of in vitro-generated platelets. We have shown that infusing megakaryocytes leads to intrapulmonary release of functional platelets. A Src kinase inhibitor (SU6656), a Rho-associated kinase inhibitor (Y27632), and an aurora B kinase inhibitor (AZD1152) have been shown to increase megakaryocyte ploidy and in vitro proplatelet release. We now tested whether megakaryocytes generated from CD34 hematopoietic cells in the presence of these inhibitors could enhance functional platelet yield following megakaryocyte infusion. As expected, all inhibitors increased megakaryocyte ploidy, size, and granularity, but these inhibitors differed in whether they injured terminal megakaryocytes: SU6656 was protective, whereas Y27632 and AZD1152 increased injury. Upon infusion, inhibitor-treated megakaryocytes released threefold to ninefold more platelets per initial noninjured megakaryocyte relative to control, but only SU6656-treated megakaryocytes had a significant increase in platelet yield when calculated based on the number of initial CD34 cells; this was fourfold over nontreated megakaryocytes. The released platelets from drug-treated, but healthy, megakaryocytes contained similar percentages of young, uninjured platelets that robustly responded to agonists and were well incorporated into a growing thrombus in vivo as controls. These studies suggest that drug screens that select megakaryocytes with enhanced ploidy, cell size, and granularity may include a subset of drugs that can enhance the yield and function of platelets, and may have clinical application for ex vivo-generated megakaryocytes and platelet transfusion.
在体外培养巨核细胞生成血小板可能有助于满足日益增长的血小板输注需求。但仍存在一些挑战，包括体外生成的血小板产量低、质量差。我们已经证明，输注巨核细胞会导致功能性血小板在肺内释放。研究表明，Src 激酶抑制剂（SU6656）、Rho 相关激酶抑制剂（Y27632）和 Aurora B 激酶抑制剂（AZD1152）可增加巨核细胞的倍性和体外产板。我们现在测试了在这些抑制剂存在的情况下，从 CD34 造血细胞生成的巨核细胞是否可以增强巨核细胞输注后功能性血小板的产量。不出所料，所有抑制剂均增加巨核细胞的倍性、大小和颗粒度，但这些抑制剂在是否损伤终末巨核细胞方面存在差异：SU6656 具有保护作用，而 Y27632 和 AZD1152 则增加损伤。输注时，与对照相比，用抑制剂处理的巨核细胞每初始未损伤巨核细胞释放的血小板增加了三倍至九倍，但仅在用 SU6656 处理的巨核细胞中，根据初始 CD34 细胞的数量计算时，血小板产量有显著增加；与未经处理的巨核细胞相比，增加了四倍。来自经药物处理但健康的巨核细胞释放的血小板含有相似比例的年轻、未受损的血小板，这些血小板对激动剂反应强烈，在体内作为对照可很好地整合到正在生长的血栓中。这些研究表明，选择具有增强的倍性、细胞大小和颗粒度的巨核细胞的药物筛选可能包括一组可增强血小板产量和功能的药物，并且可能对体外生成的巨核细胞和血小板输注具有临床应用价值。
