Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

Abstract

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. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:731-742 SIGNIFICANCEThe clinical application of human pluripotent stem cells (hPSCs) has been hindered by the lack of robust protocols able to sustain production of high cell numbers, as required for regenerative medicine. In this study, a strategy was developed for the expansion of human embryonic stem cells in welldefined culture conditions using microcarrier technology and stirred-tank bioreactors. The use of transcriptomic and metabolic tools allowed detailed characterization of the cell-based product and showed a phenotypic convergence between both hESC lines along the expansion process. This study provided valuable insights into the metabolic hallmarks of hPSC expansion and new information to guide bioprocess design and media optimization for the production of cells with higher quantity and improved quality, which are requisite for translation to the clinic.

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors

“…Accumulation of glutamate, alanine, and to some degree glycine was observed in our bioreactor processes in line with other studies [14, 48, 49]. Although consumed in our study, asparagine, isoleucine, histidine, and threonine have been described to be secreted by hESCs in microcarrier‐based bioreactor approaches [14, 49], indicating cell line and/or process‐dependent variability in hPSCs' amino acid metabolism. In CHO cells and other fast‐proliferating cells, glycine production has been associated with high rates of serine metabolism whereas glutamate and alanine accumulation was reported to indicate distinct glutamine metabolism activity [50, 51].…”

“…Applying repeated batch feeding of hESCs on matrix‐coated microcarriers in a stirred bioreactor, Silva et al recently found elevated glycolysis in 3D suspension culture compared with 2D static controls [49]. Since the same culture medium (mTeSR1) was used, the study seemingly contradicted our results.…”

Emerging strategies for the development of food industries

“…Bioprocess design is used in virtually every biological process, from surfactants and recombinant protein production to human stem cell culture [75][76][77].…”

Good Cell Culture Practice for stem cells and stem-cell-derived models

“…Additionally, "omics" technologies, such as transcriptomics, metabolomics, phospho-proteomics, and epigenomics, in combination with systems biology, while allowing the identification of the molecular mechanisms (i.e., mode of actions) underlying (toxicant) effects in a high-throughput manner, are also helping to expand current knowledge of PSC profiles, which has special relevance when PSC-derivatives are intended to be used for clinical applications (Silva et al, 2015).…”

“…One of the main advantages is its dependence on RNA and not DNA sequence (in comparison to ZFN and TALEN) as the RNA machinery base-pairing rules are simpler between an engineered RNA and the target DNA site (Sander and Joung, 2014). Meganucleases, are used for gene correction, insertion of therapeutic genes, targeted mutagenesis and virus clipping among others (Silva et al, 2015).…”