Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.

Cell stem cell (Impact Factor: 22.15). 01/2014; 14(1):13-26. DOI: 10.1016/j.stem.2013.12.005
Source: PubMed

ABSTRACT Human pluripotent stem cells (hPSCs) provide powerful resources for application in regenerative medicine and pharmaceutical development. In the past decade, various methods have been developed for large-scale hPSC culture that rely on combined use of multiple growth components, including media containing various growth factors, extracellular matrices, 3D environmental cues, and modes of multicellular association. In this Protocol Review, we dissect these growth components by comparing cell culture methods and identifying the benefits and pitfalls associated with each one. We further provide criteria, considerations, and suggestions to achieve optimal cell growth for hPSC expansion, differentiation, and use in future therapeutic applications.


Available from: Kevin G Chen, Jun 12, 2014
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    ABSTRACT: 1. Background and utility of this document In 2009 the International Stem Cell Banking Initiative (ISCBI) contributors and the Ethics Working Party of the International Stem Cell Forum published a consensus on principles of best practice for the procurement, cell banking, testing and distribution of human embryonic stem cell (hESC) lines for research purposes [1], which was broadly also applicable to human induced pluripotent stem cell (hiPSC) lines. Here, we revisit this guidance to consider what the requirements would be for delivery of the early seed stocks of stem cell lines intended for clinical applications. The term 'seed stock' is used here to describe those cryopreserved stocks of cells established early in the passage history of a pluripotent stem cell line in the lab that derived the line or a stem cell bank, hereafter called the 'repository'. The seed stocks should provide cells with suitable documentation and provenance that would enable them to be taken forward for development in human therapeutic applications. WHO recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biologicals and for the characterization of cell banks were updated in 2010 and provide a number of definitions and guiding principles that may apply to stem cells. The term 'cell bank' is used to describe a stock of vials or other containers of cells with consistent composition aliquoted from a single pool of cells of the same culture history (for other specific definitions see PAS 84 [2] and WHO [3]). Three important assumptions have been made in the preparation of this document. First, that seed stocks of hPSCs are used as starting materials to make cell banks for use in clinical trials. The cell banks made within a clinical trial would need to be established according to Good Manufacturing Practice (GMP) in a facility with a relevant product manufacturing license. These banks would need additional risk assessment focused on the new banking process/reagents and the specific intended clinical application. Second, it has been assumed that undifferenti-ated pluripotent stem cells would not be inocu-lated into patients. Third, where feeder cells are used to culture hPSC lines, their cellular nature and intimate contact with the therapeutic cells means that they should be subject to similar risk assessment and banking procedures as applied to the hPSC cells. It is important to note that responsibility for establishing and updating national regulations for medicinal products relies on National Regulatory Authorities. Therefore, national requirements for cell therapy may vary considerably. Accordingly, it is not intended that this international consensus provides comprehensive guidance that will ensure compliance with requirements in any given jurisdiction. Rather, it is designed to aid the development of clinical grade materials by providing points to consider in the preparation of seed stocks of stem cell lines for use in cell therapy. It may arise that there are circumstances where it is not reasonably possible to meet specific procedures presented in this document. Where this is the case any alternative procedures should be justified and mitigate against any adverse consequences. Finally, this document could also serve as a useful reference to assist in the evaluation of potential sources of candidate cell lines for the development of cell-based medicines, and provide the links necessary to identify some of the key differences in re gulatory requirements between countries. 2. Governance and ethics
    Regenerative Medicine 01/2015; DOI:10.2217/RME.14.93 · 3.50 Impact Factor
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    ABSTRACT: Pluripotent stem cells are a unique cell type with promising potentials in regenerative and personalized medicine. Yet the difficulty to understand and coax their seemingly stochastic differentiation and spontaneous self-renewal has largely limited their clinical applications. A call has been made by numerous researchers for a better characterization of surface proteins on these cells, in search of biomarkers that can dictate the developmental stages and lineage specifications, and can help formulate mechanistic insight of stem-cell fate choices. In the past two decades, proteomics has gained significant recognition on profiling surface proteins at high throughput. This review will summarize the impact of these studies on stem-cell biology, and discuss proteomic techniques used in these studies. A systematic comparison of all the techniques and their results is also attempted here to help reveal pros, cons, and the complementarity of the existing methods. This awareness should assist a better selection of suitable strategy for stem-cell related research, and shed light on technical improvements that can be explored in the future. This article is protected by copyright. All rights reserved.
    Proteomics 03/2015; 15(5-6). DOI:10.1002/pmic.201400300 · 3.97 Impact Factor
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    ABSTRACT: To harness the potential of human pluripotent stem cells (hPSCs), an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion). Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity) was enabled that were directly applicable to bioartificial cardiac tissue formation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    10/2014; 3(6). DOI:10.1016/j.stemcr.2014.09.017