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Sequencing Study Finds Few Rearrangements in Mouse Induced Pluripotent Stem Cells

By Andrea Anderson

NEW YORK (GenomeWeb News) – The process of reprogramming mouse cells to produce induced pluripotent stem cells does not necessarily increase the risk of DNA rearrangement or retroelement transpositions, according to a study in the latest issue of Cell Stem Cell.

Researchers from the University of Virginia and Scripps Research Institute used whole-genome, paired-end sequencing to address the question of whether iPSC production inherently leads to an increase in mutations, such as rearrangements or retroelement transpositions, and a decrease in genome stability.

Contrary to findings from past studies of human iPSC lines, the team found very few spontaneous mutations — and no retrotransposon insertions — in the three mouse iPSC lines tested.

"These results show that genome stability can persist throughout reprogramming, and argue that it is possible to generate iPSCs lacking gene-disrupting mutations using current reprogramming methods," wrote co-senior authors Kristin Baldwin of the Scripps Research Institute and Ira Hall from the University of Virginia, and their co-authors.

Additional studies are needed to determine whether it's possible to maintain the same level of genome stability in human iPSC lines, but those involved in the study are encouraged by the mouse findings.

"The jury's still out, and we'll see when the human experiments are done, but I think it's probably likely that you can find [human iPSC] lines that have essentially no genetic alterations," Hall told GenomeWeb Daily News.

The ability to generate pluripotent cells from differentiated tissue raises the possibility of being able to develop therapies for disease or damaged tissue based on iPSCs made from a patient's own cells, the researchers explained. But previous studies of iPSCs have suggested that these reprogrammed cells contain unusually high levels of copy number changes, point mutations, rearrangements, and retroelement transpositions.

If such mutations can't be avoided, it may hinder the usefulness of iPSCs, the team explained. "The biomedical utility of induced pluripotent stem cells will be diminished if most iPSC lines harbor deleterious genetic mutations."

As such, they set out to explore whether some of these mutations result from reprogramming itself or whether it might be caused by incomplete reprogramming or occur during other steps in the process.

To test this, the researchers did whole-genome paired-end sequencing with the Illumina GAII on three mouse iPSC lines, along with control cells from the mouse embryonic fetal cell population used for reprogramming. Two of the iPSC lines had been derived from the same somatic cell, while a third line came from another donor cell.

Overall, the researchers generated between 10 and 12 times physical genome coverage of each genome.

Because the genomes were sequenced to fairly low coverage over most of the genome sequence, Hall said, they did not attempt to find point mutations in the lines.

Instead, the study was designed to look specifically for rearrangements and retrotransposon insertions, which the team searched for using an algorithm known as HYDRA, which detects structural variant breakpoints and transposon insertions from paired-end mapping patterns. They also used a custom algorithm to detect structural variants from depth of coverage data.

Comparisons between the two sister iPSC lines also helped the team sort mutations present in the parental cell from those that cropped up during reprogramming. Since the original cell wasn't allowed to divide more than a couple times before the lines became separate, Hall explained, mutations shared by the sister iPSC lines are expected to have occurred in the somatic parental cell rather than reprogramming or culture processes.

Based on the data available, the researchers estimated that each reprogrammed iPSC genome harbored one to two mutations not found in parental cells.

"One thing we suspected was that even if we didn't find many copy number variants or genomic rearrangements, we thought we might find transposon activation, because when you reprogram a genome there is global, genome-wide epigenetic reprogramming," Hall noted. "It's known that when you perturb an epigenetic state that transposons can become activated."

Nevertheless, he said, there was no evidence of retroelement transpositions in the genome sequence data from the iPSC lines.

It's not clear why some genetic changes detected in past human iPSC studies were not found in the mouse iPSC lines, though the team noted that it may relate to the extent of the reprogramming in the lines or the type of somatic cells used for reprogramming.

As they reported in a past study, the researchers relied on mouse fetal fibroblasts to generate the iPSCs. These reprogrammed cells were subsequently used to produce reproductively viable mice with normal life spans, they noted, suggesting the lines were completely reprogrammed.

In humans it's not possible to do such tests of pluripotency, Hall said. But he noted that if there are situations in which human iPSC lines are incompletely reprogrammed, it could potentially account for the surplus mutations previously reported in human iPSC lines.

"If they're not completely reprogrammed, one could imagine that that might lead to instability — being sort of half way between the pluripotent and the non-pluripotent state," Hall explained. "When the cells aren't completely reprogrammed, there's potentially strong selection in culture too."

It's also too soon to exclude the possibility there are distinct iPSC mutation patterns in mouse and human iPSCs that results from species differences that affect the nature or extent of reprogramming, he added.

Hall and his team are continuing to collaborate with Baldwin and her group and plan to explore such questions over the next few years by doing extensive genome sequencing on human iPSCs made using different reprogramming protocols.

"If our results with these mouse cells are applicable to human cells, then selecting better donor cells and using more sensitive genome-survey techniques should allow us to identify reprogramming methods that can produce human iPSCs that will be safer or more useful for therapies than current lines," Baldwin said in a statement.