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Cellular aging can be monitored by DNA-methylation changes at specific CpG sites

ABSTRACT: Long-term culture associated changes need to be considered for quality control of cell preparations – especially in cellular therapy. Here we describe a simple method to track cellular aging based on continuous DNA-methylation changes at six specific CpG sites. This epigenetic signature can be used as a biomarker for various cell types to predict the state of cellular aging with regard to the number of passages or days of in vitro culture.

ORGANISM(S): Homo sapiens

PROVIDER: GSE29661 | GEO | 2012/05/22

SECONDARY ACCESSION(S): PRJNA141137

REPOSITORIES: GEO

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Project description:Mesenchymal stromal cells (MSCs) are of high relevance for the regeneration of mesenchymal tissues such as bone and cartilage. The promising role of MSCs in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age, their limited availability in human tissues and the need of in vitro expansion prior to treatment. We therefore aimed to determine to which degree in vitro aging and chronological aging may be similar processes or if in vitro culture-related changes at the cellular and molecular level are at least altered as a function of donor age. For that purpose we established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Transcriptome analysis revealed that long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function. Accordingly, functional analysis demonstrated altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging. Based on specific differences observed between the impact of chronological and in vitro MSC aging we conclude that both are distinct processes. Total mRNA obtained from MSCs from 12 month and three week old Sprague Dawley rats of passage 2, 30 and 100. Total RNA was isolated using Trizol® (Invitrogen) reagent as describes previously and purified using Qiagen RNeasy® mini kit (Qiagen, Germany, www.qiagen.com) according to manufacturers instruction. Illumina® BeadChip hybridization: Biotinylated cRNA was produced from 500ng total RNA using llumina® TotalPrep™ RNA amplification kit (Invitrogen). Illumina® RatRef-12 Expression BeadChips hybridization, washing, Cy3 streptavidin staining, and scanning were performed using Illumina® BeadStation 500 platform.

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Cellular aging can be monitored by DNA-methylation changes at specific CpG sites

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