Robust expansion of human pluripotent stem cells: integration of bioprocess design with transcriptomic and metabolomic characterization

Human embryonic stem cells (hESC) 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 in relevant quantities while maintaining their pluripotency has hindered the application of hESC and their derivatives in clinical setting. Here, we developed a scalable and well-characterized bioprocess for hESC expansion under fully-defined conditions and explored the potential of transcriptomic and metabolomic tools to evaluate the impact of culture system on hESC phenotype. Two different hESC lines (feeder-dependent and feeder-free lines) were efficiently expanded on xeno-free microcarriers in stirred culture systems. Moreover, 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 on the robustness of the cultivation process to homogenize cellular phenotype. Under low oxygen tensions, results showed a metabolic rearrangement with the up-regulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg-effect like phenotype, with no evidence of hypoxic stress response, in contrast to 2-dimensional culture. Overall, we report a scalable and fully-defined bioprocess for the propagation of hESC while guaranteeing product quality. Furthermore, the “omics” tools herein used provided relevant findings on the physiological/metabolic changes during the hESC expansion in environmentally-controlled stirred-tank bioreactors, which can contribute for more standardized production systems. 11 samples, comprising time-profile analysis of two hESC cell lines during expansion

人类胚胎干细胞（human embryonic stem cells, hESC）作为细胞替代治疗、组织工程与体外毒理学应用的理想细胞来源，具备巨大的应用潜力。然而，缺乏能够在维持其多能性的同时实现规模化扩增的标准化、稳定生物工艺，极大阻碍了hESC及其衍生产品在临床场景中的转化应用。本研究开发了一套在完全定义培养体系下可规模化扩增且经过充分表征的hESC生物工艺，并探究了转录组学与代谢组学工具用于评估培养体系对hESC表型影响的潜力。本研究选用两种不同的hESC系（饲养层依赖型与无饲养层型），在搅拌培养体系中依托无异种成分微载体实现了高效扩增。此外，两种细胞系均维持了干性标志物（如Oct-4、Nanog、SSEA-4及TRA1-60）的表达水平，且具备自发分化为三胚层的能力。全基因组转录组分析结果显示，在搅拌式生物反应器的扩增过程中，两种hESC系呈现出显著的表型趋同现象，有力证明了该培养工艺可有效实现细胞表型的均一化，具备良好的稳定性。与二维培养体系相比，低氧培养环境下的细胞发生了代谢重编程：糖酵解通路相关机制的表达上调，形成了类似瓦伯格效应的厌氧糖酵解表型，且未检测到低氧应激反应的相关信号。综上，本研究开发了一套可规模化、完全定义化的hESC扩增生物工艺，同时确保了产品质量的稳定性。此外，本研究使用的组学工具揭示了hESC在环境可控搅拌式生物反应器中扩增过程中的生理与代谢变化特征，可为构建更标准化的生产体系提供理论依据。本数据集共包含11份样本，涵盖了两种hESC系扩增过程中的时间梯度分析数据。