Researchers face challenges with stem cell lines that carry major DNA damage

Stem cells are a type of cell that can be configured to transform into almost any sort of cell in the body. They are presently used in research on organ development as well as in the initial phases of embryo development.

Scientists are increasingly turning to stem cells to create new therapies, which are recognized as cell-based therapies. Programming stem cells to develop into nerve cells to substitute those misplaced for neurodegeneration in diseases like Parkinson’s is another potential approach.

Initially, stem cells were generated from embryos, but adult skin cells can now be used to generate stem cells. These induced pluripotent stem cells (iPSCs) can now be derived from a variety of tissues, including blood, which is gaining popularity due to its ease of generation.

Scientists from the University of Cambridge and the Wellcome Sanger Institute, however, have found an issue with stem cell lines that originate from both skin cells and blood. When they analyzed the genomes of the stem cell lines in depth, they discovered that nearly three-quarters had significant DNA damage that could jeopardize their use in research and, more importantly, in cell-based therapies.

Their findings represent the major genetic study to date of iPSCs and are published in the journal Nature Genetics.

DNA is made up of three billion nucleotide pairs, which are molecules symbolized by the letters A, C, G, and T. Destruction to our DNA, such as that caused by ultraviolet radiation, can result in mutations over time, such as a letter C changing to a letter T. The “fingerprints” left on our DNA can disclose what is causing this damage. As these mutations pile up, they can have a significant impact on cell function and result in tumors.

We noticed that some of the iPS cells that we were generating looked different from each other, even when they were derived from the same patient and derived in the same experiment. The most striking thing was that pairs of iPS cells would have a vastly different genetic landscape – one line would have minimal damage and the other would have a level of mutations more commonly seen in tumors.”

Dr Foad Rouhani, University of Cambridge

“One possible reason for this could be that a cell on the surface of the skin is likely to have greater exposure to sunlight than a cell below the surface and therefore eventually may lead to iPS cells with greater levels of genomic damage,” said Dr Rouhani, who also worked at the Wellcome Sanger Institute.

The scientists used a common tactic referred to as whole genome sequencing to investigate the whole DNA of stem cell lines from various cohorts, such as the HipSci cohort at the Wellcome Sanger Institute, and found that up to 72% of the lines had major UV damage.

Almost three-quarters of the cell lines had UV damage. Some samples had an enormous amount of mutations—sometimes more than we find in tumors. We were all hugely surprised to learn this, given that most of these lines were derived from skin biopsies of healthy people.”

Serena Nik-Zainal, Professor, Medical Genetics, University of Cambridge

They decided to shift their focus away from skin-derived cell lines and instead concentrate on blood-derived iPSCs, which are becoming increasingly popular due to the ease with which blood samples can be obtained.

They discovered that while these blood-derived iPSCs had mutations, they had fewer mutations than skin-derived iPS cells and no UV damage. However, nearly a quarter of the patients had mutations in the BCOR gene, which is important in blood cancers.

They distinguished the iPSCs and transformed them into neurons, monitoring their progress as they went along to see if the BCOR mutations had any functional impact.

What we saw was that there were problems in generating neurons from iPSCs that have BCOR mutations—they tended to favor other cell types instead. This is a significant finding, particularly if one is intending to use those lines for neurological research.”

Dr Foad Rouhani, University of Cambridge

When the researchers studied the blood samples, they found that the BCOR mutations were not in the patient; rather, the procedure of culturing cells appears to raise the frequency of these mutations, which may have implications for other scientists working with cells in culture.

Researchers typically screen their cell lines for chromosomal issues, such as ensuring that the required 23 pairs of chromosomes are present. However, this would not be sufficiently clear to detect the possibly major issues identified by this new research.

Notably, without closely inspecting the genomes of these stem cells, clinicians and researchers would be uninformed of the fundamental damage that exists in the cell lines with which they are working.

“The DNA damage that we saw was at a nucleotide level. If you think of the human genome as like a book, most researchers would check the number of chapters and be satisfied that there were none missing. But what we saw was that even with the correct number of chapters in place, lots of the words were garbled,” says Professor Nik-Zainal.

Certainly, Professor Nik-Zainal says, there is a workaround: using whole genome sequencing to look for errors from the start.

“The cost of whole genome sequencing has dropped dramatically in recent years to around £500 per sample, though it's the analysis and interpretation that's the hardest bit. If a research question involves cell lines and cellular models, and particularly if we're going to introduce these lines back into patients, we may have to consider sequencing the genomes of these lines to understand what we are dealing with and get a sense of whether they are suitable for use,” says Professor Nik-Zainal.

Dr Rouhani adds: “In recent years we have been finding out more and more about how even our healthy cells carry many mutations and therefore it is not a realistic aim to produce stem cell lines with zero mutations. The goal should be to know as much as possible about the nature and extent of the DNA damage to make informed choices about the ultimate use of these stem cell lines.”

“If a line is to be used for cell-based therapies in patients for example, then we need to understand more about the implications of these mutations so that both clinicians and patients are better informed of the risks involved in the treatment.”