Stem cell research that uses pluripotent stem cells derived from human skin or blood cells has led to numerous discoveries, aided drug development, and proven useful in gene therapies. However, many of these human induced pluripotent stem cell (hiPSC) lines banked in repositories or developed in labs likely harbor thousands of undetected mutations, casting doubt on how generalizable the findings made with them can be, according to research published yesterday (August 11) in Nature Genetics. According to study coauthor and Cambridge Biomedical Research Campus medical geneticist Serena Nik-Zainal, the study reveals that the level of quality control involved in such stem cell research may not be up to snuff.
Researchers make hiPSCs by harvesting somatic cells—often from skin—from a person and then reprogramming them to enter an embryonic-like state. Nik-Zainal says that she and her colleagues were clued in to the widespread presence of mutations years ago when they noticed that multiple hiPSCs derived from the same patient appeared quite different from one another. Ultimately, they found that ultraviolet (UV) radiation, possibly linked to sunlight exposure prior to harvesting the cells, was causing significant levels of DNA damage to skin-derived hiPSCs—sometimes inducing tens of thousands of mutations. Still, the cells had been cleared for use by major repositories.
“Those ‘normal’ cell lines have as many mutations as [some] cancers,” Nik-Zainal tells The Scientist. “It was pretty striking.”
In the new study, which Nik-Zainal says began more than a decade ago, she and her colleagues ran whole genome sequencing on the stem cell lines stored and made available to researchers in two major repositories: HipSci, one of the largest iPSC banks in the world, and Insignia. The sequencing revealed that 72 percent of the skin cell–derived hiPSCs in the HipSci bank showed signs of mutations. Meanwhile, 27 percent of the blood-derived hiPSCs stored with Insignia and 18 percent of blood hiPSCs in HipSci contained multiple mutations in the BCOR gene, which is implicated in several types of cancer.
Further analysis revealed that mutations, especially the BCOR mutations in the blood-derived lines, can occur after reprogramming, meaning they didn’t originate from the human donors but rather arose as the cells replicated in the lab, likely through selective pressures the cells experience while growing and dividing in a dish.
“It’s a problem, but it’s solvable,” says Jeanne Loring, a stem cell researcher at the Scripps Research Institute who didn’t work on the study. “It’s a call that’s finally, increasingly being communicated: to analyze the cells you’re going to use by genomic analysis.”
As it stands now, scientists using these hiPSCs for research or clinical applications only need to screen and characterize their cell lines, whether they’re developed in-house or taken from a repository, to whatever extent is required by a journal or the reviewers assessing their work. In order to deposit a cell line someplace like HipSci, researchers only have to demonstrate that the stem cells don’t have any missing or duplicated chromosomes or other largescale genetic errors—analyses that would miss the myriad single-nucleotide mutations identified in the new paper.
Stem cells derived from adult somatic cells “will carry the mutational history of their past, as well as of any new mutations that occur when you are reprogramming them or growing them in culture,” Nik-Zainal says.
She and Loring both suggest that these overlooked mutations could absolutely invalidate the findings of basic or clinical studies that have already been published. But “it’s not like you’re a bad scientist if your cells acquire mutations,” says Loring. “There isn’t a way to avoid it,” except by stopping cells from dividing altogether, “which is not what you want,” she adds.
A cell line can harbor thousands of mutations and still be usable for research as long as those mutations are concentrated in irrelevant noncoding areas or don’t hit important genes, Nik-Zainal says.
Loring says that researchers need to act as a sort of proxy for the immune system, monitoring cells and pruning those with unwanted mutations. Otherwise, she says, the lines will mutate in ways that promote their own survival in culture, but that might not further whatever scientific or therapeutic purposes a researcher had in mind.
Nik-Zainal says that her work has prompted criticism from researchers in the field who seem resentful over the implication that they may need to implement whole genome sequencing as a new quality control measure. Despite being an order of magnitude less expensive than it was a decade ago, this type of sequencing remains technically complicated and difficult to perform without specialized training. However, she suggests that more thorough screenings may become more commonplace. “The day of now being able to absolutely screen using whole genomic sequence of every line is upon us,” she tells The Scientist. “We’re at an inflection point.”
Nik-Zainal says she’s already sent her findings to the UK Regenerative Medicine Platform, which supports stem cell research, among other initiatives, and that it was receptive to the issues she identified. Meanwhile, Loring says that she’s seen the issue of hiPSC mutations, which she calls “evolution in the incubator,” discussed at recent conferences and meetings.
“I think [this study] will help,” Loring says. “I think it adds strength to the argument that this kind of analysis should be done.”
Loring emphasizes that the field ought to adopt new standards, whether they’re imposed by journals, stem cell banks, or regulatory agencies—especially when it comes to using mutated hiSPCs in clinical research or gene therapies. She says she’s hopeful that cell line screening and characterization at the whole genome level will become commonplace as more researchers gain the necessary technical skills and the problem becomes more well-known.
Even if no new standards are imposed, Nik-Zainal suggests that whole-genome sequencing might be necessary for researchers who want to ensure that their research is valid: “If your whole experiment is dependent on demonstrating something in a cell line and you cannot account for the status of your cell line, that’s probably not good enough.” That’s especially true for clinical work, she says. “I would not want to have cells that had a major mutation being inserted into me—I would like to know.”
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