Pluripotent Stem Cells Robust Expansion of Human Pluripotent Stem Cells : Integration of Bioprocess DesignWith Transcriptomic and Metabolomic Characterization (original) (raw)
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Stem cells translational medicine, 2015
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 be...
npj Regenerative Medicine
The translation of laboratory-scale bioprocess protocols and technologies to industrial scales and the application of human induced pluripotent stem cell (hiPSC) derivatives in clinical trials globally presents optimism for the future of stem-cell products to impact healthcare. However, while many promising therapeutic approaches are being tested in pre-clinical studies, hiPSC-derived products currently account for a small fraction of active clinical trials. The complexity and volatility of hiPSCs present several bioprocessing challenges, where the goal is to generate a sufficiently large, high-quality, homogeneous population for downstream differentiation—the derivatives of which must retain functional efficacy and meet regulatory safety criteria in application. It is argued herein that one of the major challenges currently faced in improving the robustness of routine stem-cell biomanufacturing is in utilizing continuous, meaningful assessments of molecular and cellular characteris...
PloS one, 2016
Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will simplify the cultivation process and control variability. Here we describe the development of a robust, defined and xeno-free hPSC medium that supports reliable propagation of hPSCs and generation of human induced pluripotent stem cells (hiPSCs) from multiple somatic cell types; long-term serial subculturing of hPSCs with every-other-day (EOD) medium replacement; and banking fully characterized hPSCs. The hPSCs cultured in this medium for over 40 passages are genetically stable, retain high expression levels of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm. Im...
Progress and challenges in large scale expansion of human pluripotent stem cells
The constant supply of high cell numbers generated by defined, robust, and economically viable culture processes is indispensable for the envisioned application of human pluripotent stem cells (hPSCs) and their progenies for drug discovery and regenerative medicine. To achieve required cell numbers and to reduce process-related risks such as cell transformation, relative short batch-like production processes at industry- and clinically-relevant scale(s) must be developed and optimized. Here, we will review recent progress in the large-scale expansion of hPSCs with particular focus on suspension culture, which represents a universal strategy for controlled mass cell production. Another focus of the paper relates to bioreactor-based approaches, including technical aspects of bioreactor technologies and operation modes. Lastly, we will discuss current challenges of hPSC process engineering for enabling the transition from early stage process development to fully optimized hPSC production scale operation, a mandatory step for hPSCs’ industrial and clinical translation.
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Many attempts have been made to evaluate the safety and potency of human induced pluripotent stem cells (iPSCs) for clinical applications using transcriptome data, but results so far have been ambiguous or even contradictory. Here, we characterized stem cells at the pathway level, rather than at the gene level as has been the focus of previous work. We meta-analyzed publically-available gene expression data sets and evaluated signaling and metabolic pathway activation profiles for 20 human embryonic stem cell (ESC) lines, 12 human iPSC lines, five embryonic body lines, and six fibroblast cell lines. We demonstrated the close resemblance of iPSCs with ESCs at the pathway level, and provided examples of how pathway activity can be applied to identify iPSC line abnormalities or to predict in vitro differentiation potential. Our results indicate that pathway activation profiling is a promising strategy for evaluating the safety and potency of iPSC lines in translational medicine applica...
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Human pluripotent stem cells (hPSCs) are heralded to transform regenerative medicine and drug discovery and development, as they can potentially supply a scalable source of clinically relevant cell populations. Numerous exogenous and endogenous factors interplay to control stem cell fate; however, these are often unrecognized and unreported, undermining reproducibility and precluding the systematic analysis of hPSC response to molecular and chemical perturbations. We have developed a high-throughput (HTP) platform to screen hPSCs in configurable micro-environments, in which we optimized parameters including colony size and cell density to achieve rapid and robust cell fate responses to cues. We used this platform to perform single-cell protein expression profiling, revealing that Oct4 and Sox2 co-staining discriminate pluripotent, neuroectoderm, primitive streak, and extraembryonic cell fates. We applied this code to analyze dose responses of 27 developmental factors to delineate li...