Baffled-flow culture system enables the mass production of megakaryocytes from human embryonic stem cells by enhancing mitochondrial function

Human embryonic stem cells (hESCs) have become an ideal cell source for the ex vivo generation of megakaryocyte (MK) and platelet products for clinical applications. However, an ongoing challenge is to establish scalable culture systems to maximize the yield of stem cell-derived MKs that release platelets. We defined a specific dynamic 3D manufacturing system that could remarkably facilitate megakaryopoiesis and increase the yield of platelet-producing MKs from hESCs within a 12-day induction period. Additionally, an increased number of > 16N ploidy MKs, proplatelets, and platelets were generated from induced cells harvested on day 12 using the specific dynamic culture method. The specific dynamic culture method significantly enhanced endothelium-to-hematopoietic transition and early hematopoiesis. More importantly, MK fate was significantly facilitated in a specific dynamic manner during early hematopoiesis. Mechanistically, this dynamic culture significantly enhanced mitochondrial function via the oxidative phosphorylation pathway and caused differentiation skewing of hESCs toward megakaryopoiesis. We defined a specific dynamic culture system in a baffled-flow manner that enabled the mass production of MKs from hESCs within 12 days. This study can aid in the automatic and scalable production of MKs from stem cells using baffled-flow bioreactors and assist in the manufacturing of hESC-derived MK and platelet products. We performed RNA-Seq and assay to analyse the molecular mechanism of promoting the production of megakaryocytes from human embryonic stem cells in Baffled-flow culture system. RC were manipulated in baffled flasks with agitation. SC, as controls, were manipulated in baffled flasks without agitation.

人类胚胎干细胞（human embryonic stem cells, hESCs）已成为体外制备巨核细胞（megakaryocyte, MK）与血小板制剂用于临床应用的理想细胞来源。然而，当前仍存在一项亟待解决的核心挑战：构建可规模化的培养体系以最大化提升干细胞来源巨核细胞的产出量，从而实现血小板的有效释放。本研究确立了一种特异性动态三维培养体系，可在12天的诱导周期内显著促进巨核细胞生成，并提升hESCs来源的产血小板MK数量。此外，采用该特异性动态培养方法于第12天收获的诱导细胞，可产生更多倍性＞16N的MK、前血小板及血小板。该培养方法可显著增强内皮向造血转化与早期造血过程；更为关键的是，在早期造血阶段，MK的命运定向进程以特定动态方式得到了显著推进。机制层面，该动态培养体系可通过氧化磷酸化通路显著增强线粒体功能，并促使hESCs向巨核细胞生成方向发生分化偏向。本研究确立了一种折流式动态培养体系，可在12天内实现hESCs来源MK的规模化量产。本研究成果可为利用折流式生物反应器自动化、规模化制备干细胞来源MK提供技术支撑，同时有助于推动hESCs来源MK与血小板制剂的工业化生产。本研究通过RNA测序（RNA-Seq）与相关实验检测，分析了折流式培养体系促进人类胚胎干细胞生成MK的分子机制。其中，实验组（RC）在带搅拌的折流摇瓶中培养，对照组（SC）则在无搅拌的折流摇瓶中培养。