Researchers from Mount Sinai Hospital in Toronto, Canada, and others at the MRC Centre for Regenerative Medicine report discovering a method to create pluripotent stem cells without disrupting healthy genes. The technique uses a novel wrapping procedure to deliver specific genes to reprogram cells into an embryonic-like state instead of the usual viral-delivery mechanism.
iPS cells produced with this nonviral vector showed robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice, according to the team.
The investigators showed that nonviral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4, and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved.
When the single-vector reprogramming system was combined with a piggyBac transposon, the scientists succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers.
Investigators inserted genes that encode the Yamanaka factors —c-Myc, Klf4, Oct4 and Sox2—into a piece of DNA, or cassette, that also contained a jumping gene known as piggyBAC. This was then placed in the DNA of mouse and human skin cells and was found to reprogram the recipient cells back to an embryonic-like state.
The teams then used the enzyme transposase to remove the cassette from the mouse cells. Though some scientists are saying the technique will not count as a major advance until the cassette is removed from the human skin cells, the team is certain they will be able to use transposase to do just that. The group is currently trying to use his method to reprogram cat and dog cells.
The study appears in the March 1 edition of Nature.