Characterization and culture of human embryonic stem cells

Hoffman, Lisa; Carpenter, Melissa K.

doi:10.1038/nbt1102

Cited by 416 publications

(260 citation statements)

References 104 publications

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“…Genetic evaluation of therapeutic hESC E. Stephenson et al damage caused by reactive oxygen species and free radicals, which are generated during normal oxidative cell metabolism. Indeed, undifferentiated hESCs have a much lower expression of Cu/Zn-superoxide dismutase, glutathione peroxidase 1, and peroxiredoxin 1 and 2, which are enzymes that normally protect cells against such damage (Cho et al 2006 (Draper et al 2004;Hoffman & Carpenter 2005;Mitalipova et al 2005). Comparison of mechanical cutting of colonies and the use of enzymes for passage in the same hESC line suggested that mechanical cutting better supports maintenance of a normal karyotype in long-term culture; the enzyme-treated cell line was more likely to develop karyotype abnormalities (most often chromosome 12 and 17 trisomy).…”

Section: Extrinsic Factorsmentioning

confidence: 99%

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells

Stephenson

Ogilvie

Patel

et al. 2010

J. R. Soc. Interface.

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The use of stem cells for regenerative medicine has captured the imagination of the public, with media attention contributing to rising expectations of clinical benefits. Human embryonic stem cells (hESCs) are the best model for capital investment in stem cell therapy and there is a clear need for their robust genetic characterization before scaling-up cell expansion for that purpose. We have to be certain that the genome of the starting material is stable and normal, but the limited resolution of conventional karyotyping is unable to give us such assurance. Advanced molecular cytogenetic technologies such as array comparative genomic hybridization for identifying chromosomal imbalances, and single nucleotide polymorphism analysis for identifying ethnic background and loss of heterozygosity should be introduced as obligatory diagnostic tests for each newly derived hESC line before it is deposited in national stem cell banks. If this new quality standard becomes a requirement, as we are proposing here, it would facilitate and accelerate the banking process, since end-users would be able to select the most appropriate line for their particular application, thus improving efficiency and streamlining the route to manufacturing therapeutics. The pharmaceutical industry, which may use hESC-derived cells for drug screening, should not ignore their genomic profile as this may risk misinterpretation of results and significant waste of resources.

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Section: Extrinsic Factorsmentioning

confidence: 99%

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells

Stephenson

Ogilvie

Patel

et al. 2010

J. R. Soc. Interface.

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“…Moreover, certain sensitive cell types, such as human embryonic stem cells (hESCs) die or differentiate under restrictive metabolic conditions (7,11). To this end, Van hoof et al (7) sought to compensate for arginine to proline conversion in SILAC experiments by advocating the use of isotopically labeled arginine ( 15 N 4 -Arg) in light SILAC media as well as ( 15 N 4 -13 C 6 -Arg) in the heavy media.…”

mentioning

confidence: 99%

Prevention of Amino Acid Conversion in SILAC Experiments with Embryonic Stem Cells

Bendall

Hughes

Stewart

et al. 2008

Molecular & Cellular Proteomics

179

172

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Recent studies using stable isotope labeling with amino acids in culture (SILAC) in quantitative proteomics have made mention of the problematic conversion of isotopecoded arginine to proline in cells. The resulting converted proline peptide divides the heavy peptide ion signal causing inaccuracy when compared with the light peptide ion signal. This is of particular concern as it can effect up to half of all peptides in a proteomic experiment. Strategies to both compensate for and limit the inadvertent conversion have been demonstrated, but none have been shown to prevent it. Additionally, these methods combined with SILAC labeling in general have proven problematic in their large scale application to sensitive cell types including embryonic stem cells (ESCs) from the mouse and human. Here, we show that by providing as little as 200 mg/liter L-proline in SILAC media, the conversion of arginine to proline can be rendered completely undetectable. At the same time, there was no compromise in labeling with isotope-coded arginine, indicating there is no observable back conversion from the proline supplement. As a result, when supplemented with proline, correct interpretation of "light" and "heavy" peptide ratios could be achieved even in the worst cases of conversion. By extending these principles to ESC culture protocols and reagents we were able to routinely SILAC label both mouse and human ESCs in the absence of feeder cells and without compromising the pluripotent phenotype. This study provides the simplest protocol to prevent proline artifacts in SILAC labeling experiments with arginine. Moreover, it presents a robust, feeder cell-free, protocol for performing SILAC experiments on ESCs from both the mouse and the human.

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“…This is labor-intensive and time-consuming, and it normally generates variable cluster size and results in inconsistent cell distribution. On the other hand, enzymatic passaging dissociates the hESC colonies using enzymes such as animal-derived collagenase, dispase, and trypsin [39], xeno-free recombinant enzyme [16] or enzyme-free cell dissociation buffer [40], allowing a relatively consistent and standardized passaging of hESC. In view of clinical applications, enzymatic passaging is advantageous since it enables large-scale expansion of hESC.…”

Section: Clonal Derivation Of Hescmentioning

confidence: 99%

Xeno-free derivation and culture of human embryonic stem cells: current status, problems and challenges

et al. 2007

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npg Human embryonic stem cells (hESC) not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at present limited by xeno-contamination during the in vitro derivation and propagation of these cells. In this review, we summarize the current methodologies for the derivation and the propagation of hESC in conditions that will eventually enable the generation of clinical-grade cells for future therapeutic applications.

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Characterization and culture of human embryonic stem cells

Cited by 416 publications

References 104 publications

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells

Prevention of Amino Acid Conversion in SILAC Experiments with Embryonic Stem Cells

Xeno-free derivation and culture of human embryonic stem cells: current status, problems and challenges

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