by Monica Baker

MicroRNAs, along with transcription factors, produce homogenous iPS cell colonies from mouse fibroblasts

For the first time, microRNAs have been used to facilitate reprogramming. MicroRNAs — short stretches of nucleotides that can suppress translation of certain genes — are one of several strategies being pursued in the search for the best techniques to create induced pluripotent stem cells, a type of cell that behaves like embryonic stem cells but isn't derived from embryos and has vast implications for cell therapy, drug discovery and disease modelling. So far, all techniques to reprogram cells have required the insertion of pluripotency genes, which either directly alters a cell's DNA or creates the potential for the alteration to occur. Recently, several labs have made headway using small molecules instead of genes1. Now a team led by Robert Blelloch at the University of California, San Francisco shows that microRNAs are another potential tool for reprogramming without gene insertion2.

"I certainly think that replacing the other transcription factors, alone and in combinations, [with] miRNAs is the next logical step," says Louise Laurent of the University of California, San Diego. She notes that another group had previously used microRNAs (miRNAs) to reprogram cancer cells,3 but because they used cancer cells, it was unclear whether the strategy would work in normal cells. All this, and the small molecule work, "make it very likely that reprogramming can ultimately be achieved using relatively facile methods" that avoid any sort of genetic manipulations, says Laurent4.

Blelloch and colleagues at the University of California, San Francisco had previously identified a group of miRNAs that affected the cell cycle of embryonic stem cells, and they decided to check if these miRNAs — in particular the miR-290 cluster consisting of miR-291-3p, miR-294 and miR-295 — could facilitate reprogramming.

They used retroviruses to insert the genes for Sox2, Oct4 and Klf4, three transcription factors known to reprogram cells to pluripotency, albeit at very low rates. They did not include the gene for cMyc, a transcription factor that boosts rates of reprogramming but is associated with tumour formation. Instead, the researchers synthesized nucleotides designed to act like these miRNAs, coated them with lipids so that they could enter cells and then treated cultured mouse embryonic fibroblasts (MEFs) with these lipid miRNA mixtures at the same time as treating them with the three types of retroviruses.

Six days later, after the reprogramming process was underway, the researchers again treated the cells with the miRNAs. MiR-294 had the most potent effect, boosting the reprogramming of transduced MEFs from .01–0.05% to 0.1–0.3%. This is about three-quarters of the efficiency boost seen when cMyc is used alongside the other three transcription factors.

However, adding miR-294 alongside cMyc did not boost reprogramming rates, nor did including multiple members of the miR-290 cluster. Though cMyc does bind to the promoter of this gene cluster, the effects on the cells are not identical. cMyc causes fibroblasts to proliferate — the miR-290s do not. Also, teratomas produced from induced pluripotent stem cells reprogrammed with cMyc tend to be invasive, whereas those produced from cells reprogrammed without cMyc but with miR-294 were non-invasive.

What exactly the miRNAs do in reprogramming is unclear, but their activity seems to occur later than cMyc's first activity. In mouse fibroblasts, the miR-290 cluster is silenced by an epigenetic marker called H3K27, but in embryonic stem cells it has an activating marker (H3K4). When reprogramming with all four factors, endogenous miR-294 is activated at about the same time as other endogenous pluripotency genes like Oct4.

Blelloch is trying to see if miRNAs can replace other reprogramming factors, but he says there is "still a long ways to go" to see if that's even possible.

However, multiple miRNAs in the miR-290 cluster share a common seed sequence associated with cellular proliferation and de-differentiation. Though the research represents a useful tool for reprogramming, Blelloch says that there's a biological message as well. "These microRNAs can dramatically influence cell fates." Indeed, these reprogramming miRNAs are also upregulated in some cancers, suggesting that they may be acting to promote the less-differentiated state of malignant cancer cells.