The search for methods to reprogram somatic cells to pluripotency without permanent modification of the genome is ongoing. In a recent report, Robert Blelloch and colleagues at the University of California, San Francisco, add microRNAs to the growing list of factors that can increase the efficiency of this process (Judson et al., 2009).

Using microRNAs to reprogram cells. Reprogramming factors and microRNA mimics are delivered to fibroblasts expressing a fluorescent Oct-4 reporter; GFP-positive colonies are scored. Image courtesy of Robert Judson.

Blelloch and colleagues came to this discovery based on their prior work on mouse embryonic stem cells (mESCs). These cells are known to proliferate rapidly, which they achieve largely by bypassing a checkpoint at the G1-S transition of the cell cycle. In work on Dgcr8 knockout mESCs, in which all canonical microRNAs are lost, Blelloch and colleagues had noted that microRNAs are essential for this process. They identified a key set of microRNAs, which they named the embryonic stem cell–specific cell cycle–regulating (ESCC) microRNAs, as promoters of the unique cell cycle of mESCs.

“We think that the cell cycle and differentiation are linked,” says Blelloch. “As an [embryonic stem] cell differentiates, it simultaneously extends the G1 phase, and this extension may be absolutely required for differentiation. Conversely, if you could induce an [embryonic stem cell]-like cell cycle in a differentiated cell, you could possibly promote de-differentiation. So we thought it would make a lot of sense if these microRNAs could promote reprogramming of a somatic cell.”

Indeed, this is what the researchers saw when they examined the activity of the ESCC microRNAs in a fibroblast reprogramming assay. miR-294, as well as several other microRNAs that share the same seed sequence, increased the efficiency with which mouse embryonic fibroblasts could be reprogrammed with the transcription factors Oct-4, Sox-2 and Klf-4, by 10- to 50-fold, depending on the specific microRNA and the concentration at which it was expressed. Notably, however, microRNAs had no enhancing effect on four-factor reprogramming efficiency, when Myc was added back to the reprogramming cocktail. In addition, microRNAs could not replace either Oct-4, Sox-2 or Klf-4 in this process.

This led the researchers to think that the microRNAs might be acting downstream of Myc in promoting reprogramming, and indeed, bioinformatic analysis of existing sequence data for both Myc binding and for epigenetic marks at the promoter of the miR-290 cluster, supports this idea. Experimental testing of the possible mechanisms of microRNA function is ongoing, but Blelloch cautions that it will be difficult to tease out the function of individual microRNAs. “There are three microRNAs in the miR-290 cluster that share the same seed sequence, and that is just the beginning; there are other clusters as well, miR-17, for instance. And we know from our previous work that the system is incredibly redundant. In embryonic stem cells, we identified 11 microRNAs with the same seed sequence that were all interchangeable in their function,” he says.

Could microRNAs replace all transcription factors in the reprogramming process? Blelloch is doubtful, but points out that even replacement of some of them would be beneficial, and screens for such factors are in progress in his laboratory. Others have also reported the use of both cell-penetrating recombinant proteins—in recent work from the laboratory of Sheng Ding at the Scripps Institute (Zhou et al., 2009)—and small molecules, to achieve reprogramming. “Ultimately the goal is to use some mix of factors to replace DNA elements altogether,” Blelloch states, “and I think that will happen, but what the final cocktail will be remains to be seen.”