Human Pluripotent Stem Cell Culture: Considerations for Maintenance, Expansion, and Therapeutics

Cell stem cell (Impact Factor: 22.27). 01/2014; 14(1):13-26. DOI: 10.1016/j.stem.2013.12.005
Source: PubMed


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.

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Available from: Kevin G Chen, Jun 12, 2014
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    • "Human iPSK3 cells were derived from human foreskin fibroblasts transfected with plasmid DNA encoding reprogramming factors OCT4, NANOG, SOX2 and LIN28 (kindly provided by Dr. Stephen Duncan, Medical College of Wisconsin) [27] [28]. Human iPSK3 cells were maintained in mTeSR serum-free medium (StemCell Technologies, Inc., Vancouver, Canada) or knockout serum replacement (SR) medium supplemented with FGF-2 (40 ng/ mL) on 6-well plates coated with Geltrex (Life Technologies) [29]. The SR serum-free medium contains 80% DMEM, 20% knockout SR, 1 mM L-Glutamine, 0.1 mM b-mercaptoethanol, and 0.1 mM nonessential amino acids. "
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    ABSTRACT: Extracellular matrices (ECM) derived from pluripotent stem cells (PSCs) provide a unique tissue microenvironment that can direct cellular differentiation and tissue regeneration, and rejuvenate aged progenitor cells. The unlimited growth capacity of PSCs allows for the scalable generation of PSC-secreted ECMs. Therefore, the derivation and characterization of PSC-derived ECMs is of critical importance in drug screening, disease modeling and tissue regeneration. In this study, 3-D ECMs were generated from decellularized undifferentiated embryonic stem cell (ESC) aggregates (AGG), spontaneously differentiated embryoid bodies (EB), and ESC-derived neural progenitor cell (NPC) aggregates. The capacities of different ECMs to direct proliferation and neural differentiation of the reseeded mouse ESCs and human induced pluripotent stem cells (iPSCs) were characterized. Proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed protein expression profiles that reflected distinct niche properties for each tested ECM group. The reseeded mouse ESCs and human iPSCs responded to different types of ECMs with different cellular phenotypes. Cells grown on the AGG-ECM displayed high levels of pluripotent markers Oct-4 and Nanog, while the cells grown on the NPC-ECM showed increased expression of neural marker β-tubulin III. The expression levels of β-catenin were high for cells grown on the AGG-ECM and the EB-ECM, but reduced in cells grown on the NPC-ECM, indicating a possible role of Wnt/β-catenin signaling in the cell-matrix interactions. This study demonstrates that PSC-derived ECMs can influence stem cell fate decisions by providing a spectrum of stem cell niche microenvironments during tissue development.
    Biomaterials 09/2015; 73:231-242. DOI:10.1016/j.biomaterials.2015.09.020 · 8.56 Impact Factor
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    • "Various culture conditions have emerged with demonstrated effects in maintaining pluripotency or coaxing the directed differentiation [33] [34]. Yet, because of the lack of in-depth understanding on how these environmental factors impact stem cells, regardless the current progress no cell-expansion methods are available to provide large-quantity and high-quality homogeneous PSCs or fate-committed cells derived from PSCs that can meet the clinical needs [33] [34] "
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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 · 3.81 Impact Factor
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    • "However, in order to transform such proof-of-principle studies into viable therapeutic approaches for human TLE patients, it is critical to develop optimal human cell sources that can integrate into host circuitry and increase GABA-mediated inhibitory tone, thereby reducing seizure activity in the epileptic brain. Human pluripotent stem cell (hPSC) technologies, including induced PSCs (iPSCs), have the potential to provide an unlimited and ethically unimpeded source of therapeutic cells (Chen et al., 2014; Mallon et al., 2013; Yu et al., 2013) including human interneurons . Nevertheless, efficient translation of hPSC-derived interneurons could be hampered by their well-known, protracted maturation (Le Magueresse and Monyer, 2013; Nicholas et al., 2013). "
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    ABSTRACT: Seizure disorders debilitate more than 65,000,000 people worldwide, with temporal lobe epilepsy (TLE) being the most common form. Previous studies have shown that transplantation of GABA-releasing cells results in suppression of seizures in epileptic mice. Derivation of interneurons from human PSC has been reported, pointing to clinical translation of quality-controlled human cell sources that can enhance inhibitory drive and restore host circuitry. In this study, we demonstrate that human PSC-derived maturing GABAergic interneurons (mGIN) migrate extensively and integrate into dysfunctional circuitry of the epileptic mouse brain. Using optogenetic approaches, we find that grafted mGINs generate inhibitory postsynaptic responses in host hippocampal neurons. Importantly, even before acquiring full electrophysiological maturation, grafted neurons were capable of suppressing seizures and ameliorating behavioral abnormalities such as cognitive deficits, aggressiveness and hyperactivity. These results provide support for the potential of human PSC-derived mGIN for restorative cell therapy for epilepsy.
    Cell Stem Cell 11/2014; 15(5):559–573. DOI:10.1016/j.stem.2014.10.006 · 22.27 Impact Factor
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