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首页> 外文会议>Conference on single-use technologies II: bridging polymer science to biotechnology applications >INTENSIFYING THE MANUFACTURE OF HIPSC THERAPY PRODUCTS THROUGH METABOLIC AND PROCESS UNDERSTANDING
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INTENSIFYING THE MANUFACTURE OF HIPSC THERAPY PRODUCTS THROUGH METABOLIC AND PROCESS UNDERSTANDING

机译：通过代谢和过程认识加强HIPSC治疗产品的生产

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In vitro differentiation of human induced pluripotent stem cells into specific lineages such as cardiomyocytes (hPSC-CM) and hepatocytes (hPCS-Hep) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the implementation of protocols for hPSC expansion and differentiation, there are some technological challenges remaining include the low scalability and differentiation yields. Additionally, generated cells are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype and function. In this study, we aim to overcome this hurdle by devising bioinspired and integrated strategies to improve the generation and functionality of these hiPSC-derivatives. We also applied robust multi-parametric techniques including proteomics, transcriptomics, metabolomics and fluxomics as complementary analytical tools to support bioprocess optimization and product characterization. We cultured hiPSC as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion and used a capacitance probe for in situ monitoring of cell growth/differentiation. After cell expansion, the hepatic differentiation step was integrated by addition of key soluble factors and controlling the dissolved oxygen concentration at various stages of the process to generate populations enriched for definitive endoderm, hepatocyte progenitors and mature hepatocytes. The analyses of hepatic markers expression throughout the stages of the differentiation confirmed that hepatocyte differentiation was improved in 3D spheroids when compared to 2D culture. Noteworthy, these hiPSC-HLC exhibited functional characteristics typical of hepatocytes (albumin production, glycogen storage and CYP450 activity). We also demonstrate the potential of dielectric spectroscopy to monitor cell expansion and hepatic differentiation in STB. For CM differentiation, we relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. We assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose-and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. "-Omics" analyses showed that addition of galactose to culture medium and culturing the cells under perfusion improves total oxidative capacity of the cells and ameliorates fatty acid oxidation. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Funding: This work was supported by FCT-funded projects NETDIAMOND (SAICTPAC/0047/2015), MetaCardio (Ref.032566) and FCT/ERA-Net (ERAdicatPH; Ref. E-Rare3/0002/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged.
机译：将人诱导的多能干细胞体外分化为特定谱系，例如心肌细胞（hPSC-CM）和肝细胞（hPCS-Hep），是使其能够应用于细胞治疗和药物开发的关键过程。尽管如此，尽管在过去十年中为实现hPSC扩展和差异化协议付出了巨大的努力，但仍存在一些技术挑战，包括低可伸缩性和差异化收益。另外，在表型和功能方面，生成的细胞仍不成熟，与胎儿/胚胎细胞非常相似。在这项研究中，我们旨在通过设计受生物启发和整合的策略来改善这些hiPSC衍生物的产生和功能，从而克服这一障碍。我们还将蛋白质组学，转录组学，代谢组学和通量组学等强大的多参数技术用作辅助分析工具，以支持生物工艺优化和产品表征。我们在灌注操作的搅拌罐式生物反应器（STB）中将hiPSC培养为3D聚集体，并使用电容探针对细胞生长/分化进行原位监测。细胞扩增后，通过添加关键可溶性因子并控制过程中各个阶段的溶解氧浓度来整合肝分化步骤，以生成富集定形内胚层，肝细胞祖细胞和成熟肝细胞的种群。对整个分化阶段肝标志物表达的分析证实，与2D培养相比，3D球体的肝细胞分化得到改善。值得注意的是，这些hiPSC-HLC表现出肝细胞典型的功能特征（白蛋白产生，糖原储存和CYP450活性）。我们还展示了介电光谱技术监测机顶盒中细胞扩增和肝分化的潜力。对于CM分化，我们依靠hPSC衍生的心脏祖细胞的聚集来建立可扩展的分化方案，该方案能够产生高度纯净的CM聚集培养物。我们评估了在心脏发育过程中改变培养基成分以模仿体内底物的使用是否改善了体外hPSC-CM的进一步成熟。我们的结果表明，将hPSC-CMs从含葡萄糖的培养基转变为含半乳糖和脂肪酸的培养基，可以促进它们快速成熟，成为具有较高氧化代谢，转录特征更接近成人心室组织的转录特征，更高的肌原纤维密度和排列方式的成人状CM。 ，改善的钙离子处理，增强的收缩力以及更多的生理动作电位动力学。 “ -Omics”分析表明向培养基中添加半乳糖并在灌注下培养细胞可改善细胞的总氧化能力并改善脂肪酸氧化。这项研究表明，hPSC-CM分化/成熟过程中的代谢变化是观察到的表型和功能改变的原因，而不是结果。本文建立的基于代谢的策略由于其可扩展性，简单性和易用性而具有优于现有协议的技术和经济优势。资金：这项工作得到了FCT资助的项目NETDIAMOND（SAICTPAC / 0047/2015），MetaCardio（Ref.032566）和FCT / ERA-Net（ERAdicatPH; Ref.E-Rare3 / 0002/2015）的支持。还承认了iNOVA4Health研究部门（LISBOA-01-0145-FEDER-007344）。

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