Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general.

• MeSH
• analýza hlavních komponent MeSH
• buněčné linie MeSH
• hmotnostní spektrometrie metody   MeSH
• kalibrace MeSH
• kokultivační techniky MeSH
• lidé MeSH
• lidské embryonální kmenové buňky fyziologie   MeSH
• multivariační analýza MeSH
• myši MeSH
• neuronové sítě * MeSH
• odběr biologického vzorku MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

• MeSH
• buněčná diferenciace účinky záření   MeSH
• buněčné linie MeSH
• genetické lokusy * MeSH
• hypoxanthinfosforibosyltransferasa genetika   metabolismus   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• lidé MeSH
• mutační rychlost * MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hypoxanthinfosforibosyltransferasa MeSH

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis.

• MeSH
• antigeny CD95 genetika   metabolismus   MeSH
• apoptóza účinky léků   MeSH
• buněčná diferenciace MeSH
• embryonální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• FLIP (buněčný) antagonisté a inhibitory   genetika   metabolismus   MeSH
• harringtoniny farmakologie   MeSH
• homoharringtonin MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• kaspasa 3 genetika   metabolismus   MeSH
• kaspasa 8 genetika   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• pluripotentní kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• proliferace buněk MeSH
• protein MCL-1 genetika   metabolismus   MeSH
• protein TRAIL genetika   metabolismus   farmakologie   MeSH
• receptory TNF - typ I genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• synergismus léků MeSH
• TRAIL receptory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD95 MeSH
• FAS protein, human MeSH Prohlížeč
• FLIP (buněčný) MeSH
• harringtoniny MeSH
• homoharringtonin MeSH
• inhibitory syntézy proteinů MeSH
• kaspasa 3 MeSH
• kaspasa 8 MeSH
• malá interferující RNA MeSH
• MCL1 protein, human MeSH Prohlížeč
• protein MCL-1 MeSH
• protein TRAIL MeSH
• receptory TNF - typ I MeSH
• TNFRSF10A protein, human MeSH Prohlížeč
• TNFSF10 protein, human MeSH Prohlížeč
• TRAIL receptory MeSH