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Teams ID Genetic Glitches in Induced Pluripotent Stem Cells

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – A pair of studies appearing online today in Nature are underscoring the genetic complications that can arise in induced pluripotent stem cells.

"These mutations could alter the properties of the stem cells, affecting their applications in studying degenerative conditions and screening for drugs to treat diseases," Andras Nagy, a molecular genetics researcher affiliated with the University of Toronto and the Samuel Lunenfeld Research Institute of Mount Sinai Hospital, who was co-corresponding author on one of the new studies, said in a statement.

"In the longer term, this discovery has important implications in the use of these cells for replacement therapies in regenerative medicine," he added.

Just last month, researchers from the Salk Institute and elsewhere reported that they had detected distinct methylation patterns in iPS cells relative to embryonic stem cells and differentiated cells.

Now, the new studies indicate that these cells may carry distinct copy number changes and point mutations as well.

In the first of these studies, researchers from Canada, Finland, and Germany reported that human iPS cells — especially early-passage cells — contain copy number changes not present in the adult cells from which they were derived or in embryonic stem cells.

"Our analysis shows that these genetic changes are a result of the reprogramming process itself, which raises the concern that the resultant cell lines are mutant or defective," Nagy said.

The researchers used the Affymetrix SNP 6.0 array to assess CNVs in 22 human iPS cell lines made using fibroblast skin cells. They then compared these with CNV patterns in 17 human embryonic stem cell lines, three fibroblast lines from which iPSCs had been generated, and one unrelated skin cell line.

In general, they found, the iPSCs had about twice as many CNVs as the embryonic stem cells or adult skin cells. And the team's subsequent analyses suggest most of the mutations occur as the iPSCs are being generated, largely due to replication stress.

Indeed, they found, CNVs were larger and more common in newly generated iPSCs. Some, but not all of these mutations tapered off as the cells were passaged, with the team's experiments pointing to selection against cells with the most deleterious mutations over time.

Meanwhile, using exome sequencing, a University of California at San Diego-led group detected point mutations in all of the human iPS cell lines that they tested. Based on their analyses of specific regions of the genomes, they estimated that the lines — which had been generated from skin cells in seven different American labs using five approaches — each contain roughly six point mutations within gene-coding regions.

"Every single stem cell line we looked at had mutations," co-corresponding author Kun Zhang, a bioengineering research at UCSD, said in a statement. "[W]e expected to see ten times fewer mutations than we actually observed."

Using NimbleGen SeqCap EZ and Agilent SureSelect exome capture methods and a padlock capture method developed in house coupled with Illumina GAIIx sequencing, Kun and his colleagues sequenced protein-coding regions of 22 human iPS cell lines and nine fibroblast cell lines from which the cells were generated. The mutations detected were subsequently verified by capillary sequencing.

The researchers also used their new assay DigiQ — which relies on ultradeep sequencing to find rare mutations present at frequencies as low as 0.005 percent — to digitally quantify the mutation frequency for 32 mutations in six of the fibroblast lines prior to reprogramming.

On average, the search yielded five iPSC-specific protein-coding mutations per line tested. Based on the mutation patterns detected in the exomes, the team estimates that each iPS cell line carries around six coding mutations.

From their subsequent experiments, the researchers concluded that these changes represent mutations present in the skin cells used for reprogramming, as well as alterations that arose during and after cellular reprogramming.

"[O]ur results demonstrate that pre-existing and new mutations that occur during and after reprogramming all contribute to the high mutational load we discovered in [human iPS] cell lines," they wrote.

Most of the so-called "reprogramming-associated" point mutations detected were non-synonymous, nonsense, or splice variant changes predicted to alter the proteins produced by affected genes.

Moreover, many of the mutations fell within genes that have been implicated in cancer or Mendelian disease risk, the researchers noted. Still, they emphasized that more research is needed to understand how such changes affect cellular function, if at all.
Together, the researchers said, these and other studies point to a need for careful screening and characterization of iPSCs before these cells can reach their full potential — particularly in a clinical setting.

"[T]hings can go wrong at the genome level when reprogramming and growing reprogrammed cells," Zhang said. "So, to maximize safety, before we put these cells back in the human body for therapeutic purposes, we must be sure that the cells contain the same genome as the recipient, with no cancer-causing or other serious types of mutations."

Similarly, given his team's CNV findings, Nagy agrees that "whole genome analysis should be included as part of quality control of iPS cell lines to ensure that these cells are genetically normal after the reprogramming process."

The Scan

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