DNA methylation changes during long-term in vitro cell culture are caused by epigenetic drift.

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift.

Generation of a set of isogenic iPSC lines carrying all APOE genetic variants (Ɛ2/Ɛ3/Ɛ4) and knock-out for the study of APOE biology in health and disease.

APOE genotype is the strongest genetic risk factor for Alzheimer's Disease (AD). The low degree of homology between mouse and human APOE is a concerning issue in preclinical models currently used to study the role of this gene in AD pathophysiology. A key objective of ADAPTED (Alzheimer's Disease Apolipoprotein Pathology for Treatment Elucidation and Development) project was to generate in vitro models that better recapitulate human APOE biology. We describe a new set of induced pluripotent stem cells (iPSC) lines carrying common APOE variants (Ɛ2, Ɛ3, and Ɛ3/Ɛ4) and a knock-out isogenic to the parental APOE Ɛ4/Ɛ4 line (UKBi011-A).

Effects of senolytic drugs on human mesenchymal stromal cells.

BACKGROUND: Senolytic drugs are thought to target senescent cells and might thereby rejuvenate tissues. In fact, such compounds were suggested to increase health and lifespan in various murine aging models. So far, effects of senolytic drugs have not been analysed during replicative senescence of human mesenchymal stromal cells (MSCs). METHODS: In this study, we tested four potentially senolytic drugs: ABT-263 (navitoclax), quercetin, nicotinamide riboside, and danazol. The effects of these compounds were analysed during long-term expansion of MSCs, until replicative senescence. Furthermore, we determined the effect on molecular markers for replicative senescence, such as senescence-associated beta-galactosidase staining (SA-ß-gal), telomere attrition, and senescence-associated DNA methylation changes. RESULTS: Co-culture experiments of fluorescently labelled early and late passages revealed that particularly ABT-263 had a significant but moderate senolytic effect. This was in line with reduced SA-ß-gal staining in senescent MSCs upon treatment with ABT-263. However, none of the drugs had significant effects on the maximum number of population doublings, telomere length, or epigenetic senescence predictions. CONCLUSIONS: Of the four tested drugs, only ABT-263 revealed a senolytic effect in human MSCs-and even treatment with this compound did not rejuvenate MSCs with regard to telomere length or epigenetic senescence signature. It will be important to identify more potent senolytic drugs to meet the high hopes for regenerative medicine.

A Monoclonal Antibody That Discriminates Between SNAP-Tagged and CLIP-Tagged Proteins.

SNAP-tag technology allows recombinant proteins to be covalently labeled to O(6)-benzylguanine (BG)-modified substrates with 1:1 stoichiometry. By attaching according fluorophores, this method is ideally suited for in vitro and in vivo imaging, as well as protein interaction analyses. Fluorophores modified with BG react with the SNAP-tag, whereas those modified with O(2)-benzylcytosine (BC) conjugate to a more recent derivative known as the CLIP-tag. The orthogonal substrate specificity of the SNAP- and CLIP-tags extends the range of applications by allowing double labeling. We previously developed a monoclonal antibody (mAb) that recognizes both tags. In this study, we describe a new mAb, which is specific for the SNAP-tag alone. Therefore, this mAb allows discrimination between SNAP- and CLIP-tags within a broad range of immunological methods, including enzyme-linked immunosorbent assays, western blotting, flow cytometry, and immunohistochemistry.