Biology subjects

Besselink, N.

Publications and source records attributed to Besselink, N..

2 recordsLinked to original sources

bioRxiv2018

Mutational impact of culturing human pluripotent and adult stem cells

Genetic changes acquired during in vitro culture pose a potential risk for the successful application of stem cells in regenerative medicine. To assess mutation accumulation risks induced by culturing, we determined genetic aberrations in individual human induced pluripotent stem cells (iPS cells) and adult stem cells (ASCs) by whole genome sequencing analyses. Individual iPS cells, intestinal ASCs and liver ASCs accumulated 3.5{+/-}0.5, 7.2{+/-}1.0 and 8.4{+/-}3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of ASCs adds up to [~]1600 base pair substitutions, which is [~]40-fold higher than the in vivo rate of [~]40 base pair substitutions per year. Mutational analysis revealed a distinct in vitro induced mutational signature that is irrespective of stem cell type and distinct from the in vivo mutational signature. This in vitro signature is characterized by C to A changes that have previously been linked to oxidative stress conditions. Additionally, we observed stem cell-specific mutational signatures and differences in transcriptional strand bias, indicating differential activity of DNA repair mechanisms between stem cell types in culture. We demonstrate that the empirically defined mutation rates, spectra, and genomic distribution enable risk assessment by modelling the accumulation of specific oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Taken together, we have here for the first time accurately quantified and characterized in vitro mutation accumulation in human iPS cells and ASCs in a direct comparison. These results provide insights for further optimization of culture conditions for safe in vivo utilization of these cell types for regenerative purposes.

genomics↗

bioRxiv2017

Deficiency of global genome nucleotide excision repair explains mutational signature observed in cancer

Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging. Tumors deficient in NER are more sensitive to cisplatin treatment. Characterization of the mutational consequences of NER-deficiency may therefore provide important diagnostic opportunities. Here, we analyzed the somatic mutational profiles of adult stem cells (ASCs) from NER-deficient Ercc1-/{Delta} mice, using whole-genome sequencing analysis of clonally derived organoid cultures. Our results indicate that NER-deficiency increases the base substitution load in liver, but not in small intestinal ASCs, which coincides with a tissue-specific aging-pathology observed in these mice. The mutational landscape changes as a result of NER-deficiency in ASCs of both tissues and shows an increased contribution of Signature 8 mutations, which is a pattern with unknown etiology that is recurrently observed in various cancer types. The scattered genomic distribution of the acquired base substitutions indicates that deficiency of global-genome NER (GG-NER) is responsible for the altered mutational landscape. In line with this, we observed increased Signature 8 mutations in a GG-NER-deficient human organoid culture in which XPC was deleted using CRISPR-Cas9 gene-editing. Furthermore, genomes of NER-deficient breast tumors show an increased contribution of Signature 8 mutations compared with NER-proficient tumors. Elevated levels of Signature 8 mutations may therefore serve as a biomarker for NER-deficiency and could improve personalized cancer treatment strategies.

molecular biology↗