Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization
Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization
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DOI：
10.5966/sctm.2014-0270
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发表时间：
2015-07-01
影响因子：
6
通讯作者：
Alves, Paula M.
中科院分区：
文献类型：
--
作者：
Silva, Marta M.;Rodrigues, Ana F.;Alves, Paula M.
人类胚胎干细胞（hESC）作为细胞替代疗法、组织工程和体外毒理学应用的来源具有巨大的潜力。缺乏用于hESC扩增的标准化和稳健的生物过程阻碍了hESC及其衍生物在临床环境中的应用。我们开发了一个强大的和充分表征的生物过程中hESC扩增在完全定义的条件下，并探讨了转录组学和代谢组学工具的潜力，培养系统对细胞增殖，代谢和表型的影响进行了更全面的评估。两种不同的人胚胎干细胞系（饲养层依赖性和饲养层自由线），有效地扩大在搅拌培养系统中的无异种微载体。两种hESC系都保持了干细胞标记物如Oct-4、Nanog、SSEA-4和TRA 1 -60的表达以及自发分化成三个胚层的能力。全基因组转录组分析揭示了两种hESC系沿着搅拌罐生物反应器培养中的扩增过程的表型收敛，为培养过程的稳健性提供了强有力的证据，以均质化细胞表型。在低氧张力下，结果显示代谢重排与糖酵解机制的上调，有利于无氧糖酵解Warburg效应样表型，没有缺氧应激反应的证据，与二维培养相反。总的来说，我们报告了一个标准化的扩展生物过程，可以保证最大的产品质量。此外，所使用的“组学”工具提供了在环境控制的搅拌罐生物反应器中hESC扩增期间生理和代谢变化的相关发现，这有助于改进规模化生产系统。
Human embryonic stem cells (hESCs) have an enormous potential as a source for cell replacement therapies, tissue engineering, and in vitro toxicology applications. The lack of standardized and robust bioprocesses for hESC expansion has hindered the application of hESCs and their derivatives in clinical settings. We developed a robust and well-characterized bioprocess for hESC expansion under fully defined conditions and explored the potential of transcriptomic and metabolomic tools for a more comprehensive assessment of culture system impact on cell proliferation, metabolism, and phenotype. Two different hESC lines (feeder-dependent and feeder-free lines) were efficiently expanded on xeno-free microcarriers in stirred culture systems. Both hESC lines maintained the expression of stemness markers such as Oct-4, Nanog, SSEA-4, and TRA1-60 and the ability to spontaneously differentiate into the three germ layers. Whole-genome transcriptome profiling revealed a phenotypic convergence between both hESC lines along the expansion process in stirred-tank bioreactor cultures, providing strong evidence of the robustness of the cultivation process to homogenize cellular phenotype. Under low-oxygen tension, results showed metabolic rearrangement with upregulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg-effect-like phenotype, with no evidence of hypoxic stress response, in contrast to two-dimensional culture. Overall, we report a standardized expansion bioprocess that can guarantee maximal product quality. Furthermore, the "omics" tools used provided relevant findings on the physiological and metabolic changes during hESC expansion in environmentally controlled stirred-tank bioreactors, which can contribute to improved scale-up production systems.