Guardian of the genome goes rogue in cultured stem cells

Author: Jovana Drinjakovic, 05/10/17

Cancer-linked mutations disrupt the contact between the TP53 (blue and violet), encoded by the p53 gene and the DNA (orange), leading to DNA damage and eventually cancer. Cho et al. Science 265 pp. 346, 1994

Last month, a study published in Nature revealed that researchers have unknowingly been working on human stem cell lines that harbour mutations in a gene linked to many cancers, raising safety concerns over their use in therapy. But the findings don’t condemn stem cell treatment to an early grave. Instead, they raise an awareness of what seems to be a widespread fallout of culturing cells for a long time, and call for more rigorous tests to ensure the cells are safe before they reach patients.

Teams led by Drs. Steve McCarroll and Kevin Eggan, of the Broad Institute of Harvard and MIT, found that five percent of the examined human stem cell lines acquired mutations in a gene called p53, which is also mutated in about half of all cancers. The mutations enhanced the cells’ ability to grow and divide and they were the same as those commonly found in tumours.

“This shows that when you grow cells in vitro [in the lab dish], you are putting them under conditions that select for mutations in p53 that give them an advantage of growth, which could be dangerous if you are cultivating these cells for the purpose of transplanting them back into humans,” says Dr. Brent Derry, Senior Scientist at the Hospital for Sick Children (SickKids) and professor in the University of Toronto’s Department of Molecular Genetics. “If you do the math, this is way beyond what you would expect from mutations occurring at random,” he said.

Though worrying, the fact that mutations arise in stem cells is not surprising. Changes creep into the DNA every time the genetic material is copied during cell division. Stem cells, by their very nature, are especially prone to this because they can multiply without end, which is what makes them such a great source for spare body parts.

While the cells are pretty good at fixing errors in the DNA, this process is not foolproof and occasionally mutations remain. Most of them are harmless, but some occur in important genes and p53 is one of them.

Dubbed “guardian of the genome,” the p53 wears many hats in the cell, protecting the DNA from damage and sounding the alarm to kill off cells that begin dividing out of control. Because it normally keeps cancer at bay, the p53 is also known as a tumour suppressor.

Many genes work in a similar fashion, but to this day the p53 remains the best-known tumour suppressor, infamous for its disproportional contribution to cancer. With the p53’s protective clout taken out by mutations, the cell descends into unchecked division, picking up more DNA damage on the go in a perfect storm of molecular glitches that turn it cancerous. People who inherit p53 mutations develop Li-Fraumeni syndrome, a rare genetic condition in which a vast majority will get cancer, often in several organs.

In the study, the researchers sequenced all the genes, or the protein-coding portion of the genome, in 140 human stem cell lines, originally isolated from embryos. One hundred and fourteen of these lines are maintained by the U.S. National Institutes of Health and used by labs around the world, whereas 26 lines come from other sources and were prepared for therapeutic use by good manufacturing practice (GMP), a quality control standard set by regulatory agencies in multiple countries.

The teams looked for potentially harmful DNA changes across all genes, but the p53 stood out as the only gene with more than one such mutation present in different cell lines. The study found a total of six mutations in five cell lines, including a GMP line.

The researchers also reanalyzed previously published data on another 117 cells lines, including 104 induced pluripotent stem (iPS) lines derived from adult tissue, such as the skin, to find an additional nine p53 mutations, bringing the total to 15 mutations in 257 lines.

According to Dr. David Malkin, director of the Cancer Genetics Program at SickKids and an oncologist who discovered the link between the p53 and Li-Fraumeni syndrome, the occurrence of p53 mutations—or any other genetic changes for that matter— in cultured stem cells is not surprising and has been reported before. What is intriguing is that all discovered mutations are “pathogenic,” or well known to oncologists as the most common p53 changes seen in cancer, suggesting that the changes give the cultured cells an ability to grow faster and outnumber other cells in the dish, Dr. Malkin wrote in an e-mail.

This is supported by experimental data in the paper showing that, over the course of 40 generations in culture, the cells with p53 mutations were able to outnumber other cells thanks to their ability to grow and divide faster.

The study leaves open questions as to how the naturally occurring stem cells in the body manage to stay mutation-free. “There must be something about their niche (the environment the cells are growing in) that keeps the genome stability intact,” says Derry. Research in this area could reveal clues to cell culture conditions in which the cells do not accumulate DNA damage.

Under current regulations, the stem cells prepared for use in therapy do not have to undergo mandatory checks for mutations genome-wide, according to Dr. Jeanne Loring of the Salk Institute in La Jolla, who wrote a blog post on the study in the stem cell blog The Niche and whose lab previously reported on a spontaneous p53 mutation that spurred the growth of cultured stem cells.

No one wants to give patients cancer instead of a life-saving treatment, but this remains a grim possibility unless the cells are thoroughly checked for damaging mutations in p53 and other genes. This could be done by sequencing the cells’ genomes, which at about $2,000 far outweighs the risk of unknowingly using unsafe cells in further research and clinical trials.

Two years ago, a trailblazing study in Japan, testing the safety of iPS-derived eye tissue as a treatment for blindness, was halted when mutations were discovered in the cells that were going to be transplanted into a patient. The study has since resumed.

 

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Jovana Drinjakovic

Jovana Drinjakovic

Jovana Drinjakovic is a science writer with a background in cell and developmental biology. After completing her PhD in Cambridge (the old one) and a postdoc at the Hospital for Sick Children in Toronto, Jovana decided to switch gears and enrolled into a journalism course at the University of Toronto’s Munk School of Global Affairs. Her writing appeared in the Globe and Mail, the National Post, Dallas Morning News and U of T Magazine. Most days Jovana writes about discoveries at U of T’s Donnelly Centre, where she works as a communication specialist.
Jovana Drinjakovic

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