A single-tube real-time PCR assay for mycoplasma detection as a routine quality control of cell therapeutics

Janetzko, Karin; Rink, Gabi; Hecker, Andrea; Klueter, H.; Bugert, Peter

doi:10.1016/j.jcyt.2014.01.159

Cited by 5 publications

(6 citation statements)

References 9 publications

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“…Testing for mycoplasma contamination was performed at monthly intervals by reverse-transcriptase polymerase chain reaction (RT-PCR; ref. 5). CDDP (NeoCorp 1 mg/mL, Hexal AG) was provided by the hospital dispensary.…”

Section: Cell Lines and Reagentsmentioning

confidence: 99%

Multilayered Omics-Based Analysis of a Head and Neck Cancer Model of Cisplatin Resistance Reveals Intratumoral Heterogeneity and Treatment-Induced Clonal Selection

Niehr

Eder

Pilz

et al. 2018

Clinical Cancer Research

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Platinum-based drugs, in particular cisplatin (cis-diamminedichloridoplatinum(II), CDDP), are used for treatment of squamous cell carcinoma of the head and neck (SCCHN). Despite initial responses, CDDP treatment often results in chemoresistance, leading to therapeutic failure. The role of primary resistance at subclonal level and treatment-induced clonal selection in the development of CDDP resistance remains unknown. By applying targeted next-generation sequencing, fluorescence hybridization, microarray-based transcriptome, and mass spectrometry-based phosphoproteome analysis to the CDDP-sensitive SCCHN cell line FaDu, a CDDP-resistant subline, and single-cell derived subclones, the molecular basis of CDDP resistance was elucidated. The causal relationship between molecular features and resistant phenotypes was determined by siRNA-based gene silencing. The clinical relevance of molecular findings was validated in patients with SCCHN with recurrence after CDDP-based chemoradiation and the TCGA SCCHN dataset. Evidence of primary resistance at clonal level and clonal selection by long-term CDDP treatment was established in the FaDu model. Resistance was associated with aneuploidy of chromosome 17, increased copy-numbers and overexpression of the gain-of-function (GOF) mutant variant p53 siRNA-mediated knockdown established a causal relationship between mutant p53 and CDDP resistance. Resistant clones were also characterized by increased activity of the PI3K-AKT-mTOR pathway. The poor prognostic value of GOF variants and mTOR pathway upregulation was confirmed in the TCGA SCCHN cohort. Our study demonstrates a link of intratumoral heterogeneity and clonal evolution as important mechanisms of drug resistance in SCCHN and establishes mutant GOF variants and the PI3K/mTOR pathway as molecular targets for treatment optimization..

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Section: Cell Lines and Reagentsmentioning

confidence: 99%

Multilayered Omics-Based Analysis of a Head and Neck Cancer Model of Cisplatin Resistance Reveals Intratumoral Heterogeneity and Treatment-Induced Clonal Selection

Niehr

Eder

Pilz

et al. 2018

Clinical Cancer Research

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“…Cells were passaged as single cells using Accutase (Sigma) with Revitacell cell supplement (1/100, Invitrogen, cat#A2644501), and grew until 70% confluency, then we replated. Cultured cell lines were routinely tested for mycoplasma contamination by PCR (Janetzko et al 2014).…”

Section: Methodsmentioning

confidence: 99%

Cellular stress in brain organoids is limited to a distinct and bioinformatically removable subpopulation

Vértesy

Eichmüller

Naas

et al. 2022

Preprint

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Organoids enable disease modeling in complex and structured human tissue, in vitro. Like most 3D models, they lack sufficient oxygen supply, leading to cellular stress. These negative effects are particularly prominent in complex models, like brain organoids, where they can prevent proper lineage commitment. Here, we analyze brain organoid and fetal single cell RNA sequencing (scRNAseq) data from published and new datasets totaling over 190,000 cells. We describe a unique stress signature found in all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined organoid cell population, and present Gruffi, an algorithm that uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. Our data show that adverse effects of cell stress can be corrected by bioinformatic analysis, improving developmental trajectories and resemblance to fetal data.

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“…Cells were incubated at 37 °C, 5% CO 2 and cultured in DMEM (Gibco) with 10% Fetal Bovine Serum (VWR Life Science Seradigm) and Penicillin (5 U/ml, Sigma). All lines used in this study were certified to be free of mycoplasma by a qPCR [47] with a detection limit below 10 genomes/ml. In addition, cell lines were randomly selected for third party validation using Hoechst staining [48].…”

Section: Cell Linesmentioning

confidence: 99%

“…The authors would like to acknowledge Dr Mitch McVey for providing the PolQ null , PolQ Z2003 , BRCA KO and BRCA 47 flies, as well as Susan McMahan for technical assistance.…”

Section: Acknowledgementsmentioning

confidence: 99%

DNA polymerase theta suppresses mitotic crossing over

Carvajal-Garcia

Crown²,

Ramsden

et al. 2020

Preprint

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Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3’ single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we expand the number of homologous recombination proteins synthetic lethal with Pol θ to the Holliday junction resolvases SLX4 and GEN1. SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in very high levels of apoptosis. We observe that flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by utilizing homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.Author summaryChromosome breaks are a common threat to the stability of DNA. Mutations in genes involved in the early steps of homologous recombination (BRCA1 and BRCA2), a mostly error-free chromosome break repair pathway, lead to hereditary breast cancer. Cells lacking BRCA1 and BRCA2 rely on DNA polymerase theta, a key protein for a more error-prone pathway, for survival. Using fruit flies and mammalian cells, we have shown that mutations in genes involved in later steps of homologous recombination (SLX4 and GEN1) also make cells reliant on polymerase theta. Moreover, we have shown that polymerase theta acts upstream of a type of homologous recombination that is error-prone and depends on SLX4 and GEN1. This form of homologous recombination, termed Holliday junction resolution, creates mitotic crossovers, which can lead to loss of heterozygosity and cancer. Our results expand the cellular contexts that make cells depend on polymerase theta for survival, and the substrates that this protein can use to repair chromosome breaks.

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A single-tube real-time PCR assay for mycoplasma detection as a routine quality control of cell therapeutics

Cited by 5 publications

References 9 publications

Multilayered Omics-Based Analysis of a Head and Neck Cancer Model of Cisplatin Resistance Reveals Intratumoral Heterogeneity and Treatment-Induced Clonal Selection

Multilayered Omics-Based Analysis of a Head and Neck Cancer Model of Cisplatin Resistance Reveals Intratumoral Heterogeneity and Treatment-Induced Clonal Selection

Cellular stress in brain organoids is limited to a distinct and bioinformatically removable subpopulation

DNA polymerase theta suppresses mitotic crossing over

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