NEW YORK (GenomeWeb) – Stem cells derived from skin, blood, or clonally expanded fibroblasts should be screened for mutations in the mitochondrial genome before they are used for clinical applications, according to researchers from Oregon Health & Science University and Cincinnati Children's Hospital.
The researchers, Shoukhrat Mitalipov, director of the Center for Embryonic Cell and Gene Therapy at Oregon Health & Science University, and Taosheng Huang, a medical geneticist and director of the Mitochondrial Medicine Program at Cincinnati Children's Hospital, reported in Cell Stem Cell today that induced pluripotent stem cell lines had elevated levels of somatic mitochondrial mutations — including non-synonymous ones, and mutations in coding regions — and that the frequency of those changes increased with the age of the donor.
Stem cells derived from skin, blood, and fibroblasts are being researched for therapeutic applications due to their ability to provide an essentially limitless source of cells that are the patient's own.
However, the extent to which such cells may harbor mitochondrial DNA mutations has not been studied extensively, and in general, researchers have thought that stem cells derived from proliferating tissues, such as skin and blood, would contain few mutations, given that previous work looking at mtDNA variants in those tissues found little accumulation of variants with age.
In this study, however, the researchers sequenced the entire mitochondrial genomes of 14 adults ranging in age from 24 years to 72 years.
First, the researchers sequenced mtDNA of pooled fibroblasts from a skin biopsy containing around 1 million cells from a 72-year-old individual. In these cells, they found that although there were a few point mutations, only two were present at a high enough frequency to potentially cause pathogenic effects. However, when they generated cell lines based on 10 randomly selected fibroblasts, they found 28 mutations, four of which were present in all alleles, and nine of which were present at frequencies above 15 percent. Each individual cell line contained at least one mutation and one cell line had 12 mutations. In addition 24 of these mutations were novel, not seen in the parental fibroblast, although the researchers theorized that some had probably been present at levels too low to be detected.
The researchers then sequenced mtDNA from cell lines derived from four other individuals and observed a similar trend.
Next, they wanted to see whether mutations in the mtDNA of elderly subjects were only an issue when iPSC lines were derived from skin fibroblasts and decided to look at iPSCs generated from peripheral blood mononuclear cells. They theorized that PBMCs may not harbor as many mtDNA mutations since they have a relatively short lifespan, are continuously regenerated, and, unlike skin fibroblasts, don't require in vitro cell expansion to create an iPSC line.
The researchers first created iPSC lines from PBMCs of their original 72-year-old subject and found that these contained similar amounts of mtDNA mutations as the cell lines from skin.
When they looked at iPSC lines derived from skin and blood cells of younger individuals, they found that although the same trend of more mutations in the iPSC line than in the parental fibroblasts held true, there were fewer overall mutations as age decreased.
Finally, they took a look at the functional impact of these mutations. Out of a total of 162 variants, they found 112 in 100 different iPSCs derived from skin and 50 in 30 iPSCs derived from blood. About half the variants were above a frequency threshold associated with having a functional impact on a cell's ability to supply energy. Of those, 41 percent were non-synonymous substitutions or indels in coding regions, while another 33 percent were in genes that likely affect protein synthesis.
A mtDNA mutation database revealed that 56 percent of these variants had never been reported and 33 percent were found in less than 1 percent of the population. Five mutations had previously been linked to human disease.
The researchers then performed a functional study for mutations in the stem cell lines from two 72-year-old individuals, looking specifically at the impact on oxygen consumption of mutations thought to affect protein synthesis. They found that the stem cell lines demonstrated significantly lower activity compared to controls.
"Structural damages to the mtDNA genes in iPSCs will likely diminish their metabolic func tion in energy demanding differentiated cells and, thereby, limit their therapeutic potential and may also affect applications in disease modeling or pharmacological screening," the authors wrote.
Going forward, it will be important to "produce and screen multiple iPSC lines for chromosomal, nuclear gene, and mtDNA defects and to identify optimal tissues for iPSC induction," they added.