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Human cell lines used to model diseases have an unexpectedly ‘skewed’ or unbalanced composition of sex chromosomes, according to new research led by Alan Colman at the A*STAR Institute of Medical Biology.
Female placental mammals have two copies of the X chromosome, one inherited from the mother and the other from the father. One of these is normally silenced at random during embryonic development through a process called X chromosome inactivation (XCI).
Colman and co-workers obtained fibroblasts from healthy female controls, as well as female patients with the neurodevelopmental disorder Rett syndrome. They reprogrammed the fibroblasts into independent colonies of induced pluripotent stem cells (iPSCs), examined the status of the X chromosomes in the iPSC colonies, and found the ratio to be highly skewed. Although the original fibroblast population contained roughly equal proportions of cells expressing each of the parental X chromosomes, it appears that cell reprogramming was only possible in cells expressing one particular X chromosome.
To investigate further, the researchers isolated RNA transcripts from cultured fibroblasts every three to six cell divisions. Analysis of the RNA sequences revealed that there was a gradual shift towards the expression of the same X chromosome during division. One explanation for this is that colonies with a skewed ratio of cells expressing the X chromosome are more prone to stress, which may cause telomere shortening. Telomeres are repetitive DNA sequences at the ends of chromosomes and their length decreases with each successive cell division. When the telomere length is below a certain critical limit, the telomere will trigger cell death or senescence. The researchers suspected that fibroblasts expressing one of the X chromosomes may die prematurely, so that only those expressing the other chromosome remain in the culture.
To test this idea, the researchers expressed active and inactive forms of human telomerase reverse transcriptase (hTERT), an enzyme that lengthens telomeres, to reprogrammed fibroblasts. They found that fibroblast populations expressing active hTERT contained equal proportions of both X chromosomes, suggesting that the enzyme attenuates or prevents the skewing.
The findings suggest that skewed X chromosome expression may be a hitherto unrecognized variable in studies using iPSCs, including those for studying Rett Syndrome and diseases linked to genes located on the X chromosome.
“It is estimated that at least 20% of genes on the two X chromosomes differ significantly,” says Colman. “Such differences might influence a cell’s ability to deal with various environmental stresses, such as DNA damage, and we are performing a set of experiments to test this.” The researchers note that the study also has important implications for the interpretation of long-term culture studies in cancer and senescence.
The A*STAR-affiliated researchers contributing to this research are from the Institute of Medical Biology.
