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Vector-Triggered Innate Immune Activity Is Necessary for iPSCs

By Anette Breindl
Science Editor

Researchers reported last week that the retroviral vector that is used for gene delivery in the generation of induced pluripotent stem cells, or iPSCs, does more than just ferry its cargo into cells. It activates the innate immune system – and that activation is critical for efficient reprogramming.

Senior author John Cooke said he hopes that by learning more about the process, which he and his team have termed "transflammation," transforming cells via proteins or small molecules rather than by genetic means will reach the sort of efficiency that can make the technique clinically relevant.

But more basically, he told BioWorld Today, "the importance of this paper is not that we found a better way to reprogram." It is that "we've found a way of enhancing a cell's plasticity and fluidity."

He said his team's findings could pave the way to direct reprogramming from one cell type to another, without the need for a pluripotent intermediate – which always bears some risk of going off in a different direction than its maker intended.

Such treatments could be used in vivo, for example after injuries, to turn fibroblasts into useful cell types rather than the scar tissue they now form. "What if we could heal with tissue instead of with scars? It's not so far-fetched," he said, pointing out that is the way lower vertebrates heal their injuries.

Two teams first reported in 2007 that they had managed to generate cells whose plasticity in many respects rivaled that of embryonic stem cells by transforming them with a cocktail of four transcription factors. Their work won the Nobel Prize earlier this year. (See BioWorld Today, Nov. 21, 2007, and Oct 10, 2012.)

Since that time, one of the goals for getting the technique clinic-ready has been to get rid of the viral vectors used in the transformation. Such efforts have succeeded in principle. But to date, the ability of using cell-permeant proteins, or CPPs, has not come within striking distance of viral vectors. While reprogramming with viral vectors can transform up to 1 in 100 fibroblasts that are exposed to them, the rate with proteins is in the range of 1 in 100,000.

Cooke, who is at Stanford University, described his own team's attempts to reprogram cells using CPPs. "We struggled. We were not able to get reprogramming to any reasonably efficiency."

When using CPPs rather than viral vectors, the resulting transformation process could be summed up as "low and slow," he said. While viral vector delivery leads to a rise in protein levels within hours of transfection, with CPPs, "we saw nothing on day one, we saw nothing on day two. We started to see a little hint of something on day three." But even after the process finally got started, protein levels never rose to anywhere near those in virally transformed cells.

The most obvious difference between the methods, Cooke said, was the viral vector that the team was trying to shed. "I thought, 'Maybe the virus is doing something.' So I asked [co-first author] Jieun Lee to throw in an irrelevant virus."

That strategy turned out to be what it took to get the transformation to take. When the team added a retroviral vector that was not coding for the transcription factors to protein forms of either Sox-2 or Oct-4, which are both ingredients of the iPSC transformation cocktail, their levels rose more rapidly, and to final concentrations that resembled those that result from delivering their genes via a viral vector.

Lee, her co-first author Nazish Sayed and their colleagues went on to investigate how the vector was jumpstarting the effects of the transcription factors. They found that the innate immune system, via its TLR3 receptor, responded to the viral vector. Ultimately, the TLR3 activation affected several of the cell's epigenetic markers, changing them in a way that readies the DNA at those markers accessible to the cell's translation machinery. They published their findings in the Oct. 26, 2012, issue of Cell.

Cooke said he thinks that opening up of the cell's chromatin is a way for cells to cope with infection. "Human cells recognize that the pathogen is at the door," he said, "and the alarms go off, and the cell opens up its genetic toolbox."

He added that his idea for why innate immune system activation should induce an open state in the cellular chromatin is "speculation. But it makes the most sense."

If such plasticity and fluidity can be harnessed where it is wanted, it must also be considered where it is not. The work of Cooke and his team also showed that vectors are not mere delivery vehicles, but instead affect the fate of their cargo via their effects on the cells to which they deliver that cargo. "Any time anyone uses a retroviral vector," Cooke said, "they will have to take into account the epigenetics of the cell."