Laboratory of Stem Cells and Translational Medicine, Institute for Clinical Medicine, the Second Affiliation Hospital, School of Medicine, South China University of Technology, No.1 Panfu Road, Guangzhou, 510180, People's Republic of China.
Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou, 510006, China.
Stem Cell Res Ther. 2024 Nov 28;15(1):454. doi: 10.1186/s13287-024-04071-x.
Platelet transfusion therapy has made a great breakthrough in clinical practice, and the differentiation of human embryonic stem cells (hESCs) to produce functional platelets has become a new potential approach, however, efficient generation of functional platelets still faces great challenges. Here, we presented a novel approach to highly and efficiently generate mature megakaryocytes (MKs) and functional platelets from hESCs.
In hypoxic conditions, we successfully replicated the maturation process of MKs and platelets in a controlled in vitro environment by introducing an optimal combination of cytokines at various stages of development. This method led to the generation of MKs and platelets derived from hESCs. Subsequently, mature MKs and functional platelets were further comprehensively investigated and characterized using a variety of methodologies, including flow cytometry analysis, RT-qPCR validation, Giemsa-Wright's staining, immunofluorescent staining, RNA transcriptome analysis, and DNA ploidy analysis. Additionally, the in vivo function of platelets was evaluated through the transplantation using thrombocytopenia model mice.
Under our 3D differentiation conditions with four sequential stages, hESCs could be efficiently induced into mature MKs, with 95% expressing CD41aCD42a or 90% expressing CD41aCD42b, and those MKs exhibited polyploid properties, produced filamentous proplatelet structures and further generated platelets. Furthermore, 95% of platelets showed CD42bCD62p phenotype upon the stimulation with ADP and TRAP-6, while 50% of platelets exhibited the ability to bind PAC-1, indicating that hESC-derived platelets possessed the in vitro functionality. In mice models of thrombocytopenia, hESC-derived platelets effectively restored hemostasis in a manner comparable to human blood-derived platelets. Further investigation on the mechanism of this sequential differentiation revealed that cellular differentiation and molecular interactions during the generation of hESC-derived MKs and platelets recapitulated the developmental trajectory of the megakaryopoiesis and thrombopoiesis.
Thus, our results demonstrated that we successfully established a highly efficient differentiation of hESCs into mature MKs and functional platelets in vitro. The in vivo functionality of hESC-derived platelets closely resembles that of natural human platelets, thus offering a promising avenue for the development of functional platelets suitable for future clinical applications.
血小板输血治疗在临床实践中取得了重大突破，人类胚胎干细胞（hESC）分化为功能性血小板成为一种新的潜在方法，然而，高效产生功能性血小板仍然面临巨大挑战。在这里，我们提出了一种从 hESC 高效产生成熟巨核细胞（MK）和功能性血小板的新方法。
在低氧条件下，我们通过在不同发育阶段引入最佳组合的细胞因子，在可控的体外环境中成功复制了 MK 和血小板的成熟过程。该方法导致 hESC 来源的 MK 和血小板的产生。随后，使用多种方法进一步全面研究和表征成熟的 MK 和功能性血小板，包括流式细胞术分析、RT-qPCR 验证、吉姆萨-赖特染色、免疫荧光染色、RNA 转录组分析和 DNA 倍性分析。此外，通过使用血小板减少症模型小鼠进行移植来评估血小板的体内功能。
在我们具有四个连续阶段的 3D 分化条件下，hESC 可以高效诱导为成熟的 MK，其中 95%表达 CD41aCD42a 或 90%表达 CD41aCD42b，并且这些 MK 表现出多倍体特性，产生丝状原血小板结构并进一步产生血小板。此外，在 ADP 和 TRAP-6 的刺激下，95%的血小板表现出 CD42bCD62p 表型，而 50%的血小板表现出结合 PAC-1 的能力，表明 hESC 衍生的血小板具有体外功能。在血小板减少症的小鼠模型中，hESC 衍生的血小板有效地恢复了止血作用，与人类血液衍生的血小板相当。对这种顺序分化的机制的进一步研究表明，hESC 衍生的 MK 和血小板生成过程中的细胞分化和分子相互作用再现了巨核细胞生成和血小板生成的发育轨迹。
因此，我们的结果表明，我们成功地在体外建立了一种高效地从 hESC 分化为成熟的 MK 和功能性血小板的方法。hESC 衍生血小板的体内功能与天然人类血小板非常相似，为未来临床应用中功能性血小板的开发提供了有前途的途径。
