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Mutations, Methylation in Induced Pluripotent Stem Cells Increase With Age

NEW YORK (GenomeWeb) – Induced pluripotent stem cells (iPSCs) generated with samples from older individuals contain more somatic mutations than those from younger individuals, maintaining at least some age-related methylation signatures despite reprogramming, a new study suggests.

"Any time a cell divides, there is a risk of a mutation occurring. Over time, those risks multiply," co-senior author Ali Torkamani, director of genome informatics at the Scripps Translational Science Institute, said in a statement. "Our study highlights that increased risk of mutations in iPSCs made from older donors of source cells."

As they reported in Nature Biotechnology, Torkamani, Scripps neuroscience researcher Kristin Baldwin, and their colleagues generated iPSCs from 15 individuals: five young people under 30 years old, five middle-aged individuals 49- to 79-years-old, and five Wellderly Study participants, an elderly group that included individuals between 86 and 100 years old.

Using array-based methylation profiling and exome sequencing on the stem cells, the team found that somatic mutation rates in iPSC lines rose with increasing age in the individuals, as did persistence of age-related cytosine methylation marks. On the other hand, age-related mutations appeared to level off or decline in iPSC cells generated from individuals over 90 years old, potentially due to somewhat lower blood cell progenitor divisions than anticipated in this age group.

Moreover, the study's authors noted that "extended passaging can erase most of the residual aberrant DNA methylation, indicating that improved reprogramming techniques or increased monitoring of methylation could improve iPSC safety and quality."

Although iPSCs appear to be a promising avenue for cell replacement therapies, the team noted, the full suite of factors influencing iPSC quality and subsequent cellular differentiation — including the age of individuals donating somatic tissue for iPSC generation — have yet to be characterized completely. Such considerations are particularly important for methods that employ iPSCs produced from an individual's own somatic tissue to avoid immune responses or the need for immunosuppression.

"Using iPSCs for treatment has already been initiated in Japan in a woman with age-related macular degeneration," co-author Eric Topol, a genomics researcher at the Scripps Translational Science Institute, said in a statement. "Accordingly, it's vital that we fully understand the effects of aging on these cells being cultivated to treat patients in the future."

For their analysis, the researchers used Illumina HiSeq 2500 instruments to sequence protein-coding sequences captured with Agilent SureSelect technology from each individuals' blood cell samples and from three iPSC lines apiece per person. They also used Illumina Infinium arrays to profile cytosine methylation patterns in the cells before and after reprogramming.

Consistent with past studies, the team tracked down cytosine methylation signatures in the peripheral blood mononuclear cell samples that corresponded with individuals' age. But even after reprogramming, the methylation at these sites was significantly higher in iPSC cells from older individuals than that detected in those from younger individuals.

In particular, the researchers focused on 3,896 cytosine methylation sites that are typically de-methylated during reprogramming to generate iPSCs. Methylation remained slightly higher at these sites in iPSCs from older individuals, they reported, with global methylation levels remaining roughly 5 percent higher in iPSCs from the elderly.

At almost three-dozen sites that appeared especially prone to persistent methylation in elderly iPSCs, the team noted that the process of additional cell passaging eventually led to iPSCs with more youthful methylation levels at 28 of the sites.

On the somatic mutation side, meanwhile, the researchers saw roughly twice as many somatic mutations in iPSCs from 90-year-old individuals compared to iPSCs generated from 20 year olds, reflecting the general relationship between mutation and aging. That relationship fell off in the over-90-year-old group — a shift that may be due to declining hematopoietic stem cell pools in the longest-lived individuals.

"This work highlights some considerations for clinical translation of reprogramming techniques to the clinic and offers an approach for investigating the impact of age on the epigenetic and genetic features of iPSCs and somatic cells," the authors concluded.