Extracellular vesicle biogenesis of three‐dimensional human pluripotent stem cells in a novel Vertical‐Wheel bioreactor
Extracellular vesicle biogenesis of three‐dimensional human pluripotent stem cells in a novel Vertical‐Wheel bioreactor
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DOI：
10.1002/jex2.133
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发表时间：
2024-01
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通讯作者：
Laureana Muok;Li Sun;Colin Esmonde;Hannah Worden;Cynthia Vied;Leanne Duke;Shaoyang Ma;Olivia Z Zeng;Tristan Driscoll;Sunghoon Jung;Yan Li
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作者：
Laureana Muok;Li Sun;Colin Esmonde;Hannah Worden;Cynthia Vied;Leanne Duke;Shaoyang Ma;Olivia Z Zeng;Tristan Driscoll;Sunghoon Jung;Yan Li
人诱导多能干细胞（hipsc）分泌的细胞外囊泡（EVs）在多种疾病的无细胞治疗中具有巨大的潜力，包括预防血脑屏障衰老和中风。然而，由于需要大量大规模生产，hiPSC - ev的临床前和临床应用仍然存在挑战。垂直轮式生物反应器（vwbr）具有设计特点，允许在低剪切应力下使用可扩展的聚集体或基于微载体的培养系统进行hiPSC - ev的生物制造。研究了未分化的hiPSCs在vwbr中以3 - D聚集体和Synthemax II微载体上分泌EV的情况。此外，比较了两种EV收集介质mTeSR和HBM。通过代谢物和转录组分析以及EV生物发生标记对hiPSCs进行了表征。蛋白质组学和microRNA - seq分别分析蛋白质和microRNA货物。体外进行小胶质细胞刺激和增殖功能测定。hipsc扩增为3‐D聚集体，在微载体上具有相当的细胞数量，而微载体培养具有更高的葡萄糖消耗，更高的糖酵解和更低的自噬基因表达（基于mRNA‐seq）。微载体培养的EV分泌量至少比HBM培养基高17-23倍，在mTeSR中收集EV的产量比HBM培养基高2.7-3.7倍。与其他组相比，mTeSR EV收集的微载体培养具有更小的EV大小，并且货物富含减少凋亡和促进细胞增殖的蛋白质（蛋白质组学）和mirna (microRNA‐seq)（例如Wnt‐相关途径）。在体外实验中，hiPSC‐ev显示出刺激小胶质细胞增殖和M2极化的能力。HiPSC在微载体上的扩展比在vwbr中的聚集产生更高的ev产率。与HBM相比，mTeSR中的EV收集提高了产量。mTeSR中微载体培养的生物制造ev具有外泌体特征，并具有刺激小胶质细胞的功能，这为未来的体内抗衰老研究铺平了道路。
Abstract Extracellular vesicles (EVs) secreted by human‐induced pluripotent stem cells (hiPSCs) have great potential as cell‐free therapies in various diseases, including prevention of blood–brain barrier senescence and stroke. However, there are still challenges in pre‐clinical and clinical use of hiPSC‐EVs due to the need for large‐scale production of a large quantity. Vertical‐Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC‐EVs using a scalable aggregate or microcarrier‐based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3‐D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA‐seq, respectively. The in vitro functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3‐D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA‐seq. The microcarrier cultures had at least 17–23 fold higher EV secretion, and EV collection in mTeSR had 2.7–3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA‐seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt‐related pathways). hiPSC‐EVs demonstrated the ability of stimulating proliferation and M2 polarization of microglia in vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti‐aging study.