Epigenetic drift during long-term culture of cells <i>in vitro</i>

Franzen, Julia; Georgomanolis, Theodoros; Selich, Anton; Kuo, Chao-Chung; Stöger, Reinhard; Brant, Lilija; Mulabdić, Melita Sara; Fernández-Rebollo, Eduardo; Grezella, Clara; Ostrowska, Alina; Begemann, Matthias; Nikolić, Miloš; Rath, Björn; Ho, Anthony D.; Rothe, Michael; Schambach, Axel; Papantonis, Argyris; Wagner, Wolfgang

doi:10.1101/445114

Cited by 6 publications

(9 citation statements)

References 75 publications

(82 reference statements)

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“…Furthermore, our analysis of DNAm patterns in individual reads of BBA-seq demonstrated that neighboring CpGs are modified rather independently. We have recently described similar findings for DNAm changes during long-term culture of cells in vitro (Franzen et al 2017;Franzen et al 2018). While the stochastic changes at neighboring CpGs challenge the view of directed regulation of age-associated DNAm, they may support the notion that this process is evoked by "epigenetic drift", possibly caused by changes in chromatin conformation.…”

Section: Discussionsupporting

confidence: 71%

New Targeted Approaches for Epigenetic Age Predictions

Han

Franzen

Stiehl

et al. 2019

Preprint

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Aging causes epigenetic modifications, which are utilized as a biomarker for the aging process.While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective highthroughput analysis. In this study, we utilized 4,650 Illumina BeadChip datasets of blood to select the best suited CpG sites for targeted analysis. DNA methylation analysis at these sites with either pyrosequencing or droplet digital PCR (ddPCR) revealed a high correlation with chronological age.In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower precision at individual CpGs. However, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often accompanied by a CTCF binding site at the peak. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified but reveals a stochastic pattern. Based on this, we have developed an alternative model for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. Thus, the stochastic evolution of age-associated DNA methylation patterns, which seems to resemble epigenetic drift, enables epigenetic clocks for individual DNA strands.

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Section: Discussionsupporting

confidence: 71%

New Targeted Approaches for Epigenetic Age Predictions

Han

Franzen

Stiehl

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Furthermore, our analysis of DNAm patterns in individual reads of BBA-seq demonstrated that neighboring CpGs are modified rather independently. We have recently described similar findings for DNAm changes during long-term culture of cells in vitro [29,60]. While the stochastic changes at neighboring CpGs challenge the view of directed regulation of age-associated DNAm, they may support the notion that this process is evoked by "epigenetic drift," possibly caused by changes in chromatin conformation.…”

Section: Discussionsupporting

confidence: 62%

New targeted approaches for epigenetic age predictions

et al. 2020

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Background: Age-associated DNA methylation changes provide a promising biomarker for the aging process. While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective high-throughput analysis. Results: In this study, we utilized 4647 Illumina BeadChip profiles of blood to select CpG sites that facilitate reliable age-predictions based on pyrosequencing. We demonstrate that the precision of DNA methylation measurements can be further increased with droplet digital PCR (ddPCR). In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower correlation between chronological age and DNA methylation at individual CpGs, while the age-predictions were overall relatively accurate. Furthermore, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often associated with a CTCF binding site. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified, but reveals a stochastic pattern. Based on this, we have developed a new approach for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. Conclusion: Targeted DNA methylation analysis at few age-associated CpGs by pyrosequencing, BBA-seq, and particularly ddPCR enables high precision of epigenetic age-predictions. Furthermore, we demonstrate that the stochastic evolution of age-associated DNA methylation patterns in BBA-seq data enables epigenetic clocks for individual DNA strands.

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“…Various genomic regions become continuously hyper-or hypomethylated during culture expansion [14,15]. We have previously shown that these modifications are independently acquired at neighboring CG dinucleotides (CpGs) [16,17]. A predominant DNA methylation pattern may, therefore, reveal the prevalence of dominant subclones [18].…”

Section: Introductionmentioning

confidence: 99%

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells

et al. 2020

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Background: The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion. Methods: In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays. Results: Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs. Conclusions: Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

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Epigenetic drift during long-term culture of cells in vitro

Cited by 6 publications

References 75 publications

New Targeted Approaches for Epigenetic Age Predictions

New Targeted Approaches for Epigenetic Age Predictions

New targeted approaches for epigenetic age predictions

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells

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