Highly efficient generation of mature megakaryocytes and functional platelets from human embryonic stem cells.

Background: 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.
Methods: 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.
Results: 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 CD42b ⁺ CD62p ⁺ 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.
Conclusions: 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.
Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Ethics Committee of Guangzhou First people's Hospital (Approval No.: K-2021–008-01, and approval date: January 17, 2022.) to use umbilical cord blood and human platelets from healthy volunteers for Large-scale Preparation of Functional Platelets from hESCs/iPSCs. The patients or their guardians/legally authorized representatives provided written informed consent for participation in the study with the use of umbilical cord blood and human platelets. All mouse experiments were performed according to our experimental protocols approved by the Guangzhou Committee for the Use and Care of Laboratory Animals, and by the Animal Ethics Committee of South China University of Technology University (Approval number: 2019073, and approval date: December 12, 2019.). WiCell Research Institute has confirmed that there was the ethical approval for collection of human embryonic stem cells (WA01(H1) and WA09(H9)) with NIH Approval NIHhESC-10–0043 and NIH Approval NIHhESC-10–0062, and that the donors had signed informed consent ( www.wicell.org ). Competing  interests: The authors declare that there is no competing  interests.
(© 2024. The Author(s).)