Cortin Valérie, Pineault Nicolas, Garnier Alain
Département de Recherche et Développement, Héma-Québec, Québec City, Québec, Canada.
Methods Mol Biol. 2009;482:109-26. doi: 10.1007/978-1-59745-060-7_7.
The identification and cloning of thrombopoietin was certainly a defining moment for the study of megakaryopoiesis and thrombopoiesis ex vivo. This and other progresses made in the development of culture processes for hematopoietic stem cells have paved the way for ongoing clinical trials and, in the future, for the potential therapeutic use of ex vivo produced blood substitutes such as platelets. This chapter describes a 14-day culture protocol for the production of megakaryocytes (MK) and platelets from human cord blood stem cells. The CD34+ cells are grown in a serum-free medium supplemented with a newly developed cytokine cocktail optimizing MK differentiation, expansion, and maturation. A detailed methodology for flow cytometry analysis of the cells and platelets is also presented together with supporting figures. A brief review on megakaryocytic differentiation and ex vivo MK cultures is first presented.
血小板生成素的鉴定与克隆无疑是体外巨核细胞生成和血小板生成研究的一个决定性时刻。造血干细胞培养方法的这一进展以及其他进展为正在进行的临床试验铺平了道路，并在未来为体外生产的血液替代品（如血小板）的潜在治疗应用奠定了基础。本章描述了一种从人脐带血干细胞生产巨核细胞（MK）和血小板的14天培养方案。CD34+细胞在一种无血清培养基中生长，该培养基添加了一种新开发的细胞因子混合物，可优化MK的分化、扩增和成熟。还介绍了细胞和血小板流式细胞术分析的详细方法以及辅助图表。首先简要回顾一下巨核细胞分化和体外MK培养。
