To see how serious a problem CNVs might pose for iPS cells' use, the collaborators performed tiny skin biopsies on seven volunteers and extracted cells called fibroblasts, which abound in skin and are amenable to cell culture in general and iPS cell generation in particular. From these, the team produced 20 separate iPS cell lines in culture. Using now-standard lab methods, the investigators determined, chemical unit by chemical unit, the full genomic sequence of the cells composing each new iPS cell line.
Urban and his colleagues, who had likewise assessed the fibroblasts from which the lines were derived, compared their genomic sequences with those of the newly generated iPS cells. The scientists were able to pinpoint numerous CNVs in the new cells that hadn't shown up in the fibroblasts. This raised the possibility that the rigors of reprogramming or life in a dish, or both, had led to new CNVs in the cells.
But the technique used to determine the full-length genome sequences of iPS cells and the "parental" fibroblasts from which they'd been spawned analyzes not single cells but millions at a time. So a CNV residing in only a minority of cells within this mix could easily be missed, its signal swamped by the noise of the majority report.
Armed with knowledge of the precise locations along the genome where each of the "new" CNVs had popped up in their iPS cell lines, the scientists went back to the fibroblasts. This time, they used an analytical tool that, like a photocopying machine, can generate millions or billions of copies of a single section of DNA provided that the specific DNA section is present to begin with. For each "new" CNV that had been unearthed in the iPS cells, a different version of this molecular copying machine was employed.
"Lo and behold, in many cases, this technique unearthed CNVs in the fibroblasts
|Contact: Bruce Goldman|
Stanford University Medical Center