A Faster Reset Button for Stem Cells

Being able to restore function to organs damaged through disease or injury is a goal that has not only inspired work on programming robots to act on our thoughts (see ‘We have the technology…’) but has also stimulated scientists to explore whether our bodies’ cells can be reprogrammed to take on the missing functions. The 2012 Nobel Prize committee recognised the potential for therapy in adult cell reprogramming and rewarded John Gurdon and Shinya Yamanaka for their pioneering work in this area. These two stem cell biologists realised that a key step in the process is to return the adult cells to a primitive state before persuading them to take on another role, and that this would involve resetting the cells’ nuclear programme.

Shinya Yamanaka published his nobel prize winning work in 2006, in which he showed that treating fully developed adult cells with four protein factors would drive the necessary changes in the cells’ nucleus to return them to a stem cell-like state called pluripotency; and hence the name, “induced pluripotent stem cells”, iPS cells for short.  Since then teams the world over have sought to tweak and streamline this promising technology to obtain greater numbers of pluripotent stem cells, in shorter time, from adult tissue.

Austin Smith and colleagues at the Stem Cell Institute in Cambridge, UK asked whether adult stem cells, such as neural stem cells, which are naturally available in small numbers in adult organs, could be more rapidly and efficiently converted to a state of pluripotency than their fully differentiated neuronal neighbours. As published in their PLOS Biology article in 2008, the answer turned out to be yes – neural stem cells are easier to reprogram to a pluripotent state and therefore better starting material than mature fully developed adult brain cells are! The team also made some important discoveries along the way.

One such key discovery was that these neural stem cells don’t return to the pluripotent state in a single step. Using the reprogramming treatment identified by Yamanaka’s lab (the use of viral vectors to introduce genes encoding four reprogramming factors), Smith and his lab found that neural stem cells showed signs of reprogramming much earlier (3 days versus 3 weeks) and at higher frequency than did fully differentiated cells. There was a problem, however, in that these early appearing cells arrested on the verge of full pluripotency.

José Silva, the first author of the PLOS Biology article, told me of his initial disappointment “My passion has been the study of the biology of nuclear reprogramming for many years now. When Kazutoshi Takahashi and Shinya Yamanaka published their seminal work on induced pluripotency, my imagination ran loose with ideas. The irony is that it was the initial failure to generate Induced Pluripotent Stem cells using the conventional Yamanaka factors and traditional Embryonic Stem Cell culture conditions that guided the creation of the PLoS biology work. All we could make initially were highly proliferative cells that looked like Embryonic Stem Cells but were not like these at the molecular level. Somehow these cells were not able to go all the way.”

To tackle this issue, the team broke down the differentiation process  into several steps – starting from embryonic stem cell, and ending with neural stem cell (embryonic stem cells can form any cell type in the body, whereas neural stem cells are restricted to forming only nerve cells). They then worked out which signals might be getting in the way of complete reprogramming. Using chemicals to neutralize these signals (inhibitors ERK and GSK – termed “2i”) and adding a factor called LIF, which encourages self- renewal, the team found that this chemical cocktail (called 2i/LIF) could push the early appearing, partially reprogrammed cells to adopt a fully pluripotent state. 

José Silva explains: ‘we discovered the importance of the culture environment, together with the Yamanaka factors, in instructing the conversion of a differentiated cell back into an embryonic stem cell. “

That the group were able  to generate greater numbers of iPS cells is down to their recognising the potential in these partially reprogrammed “pre-iPS” cells which may have been  previously dismissed by others;  as Kathrin Plath and colleagues at UCLA wrote in 2012, ‘While it is not absolutely clear that pre-iPS cells represent an intermediate that occurs transiently during the reprogramming process, they are not simply an aborted reprogramming artifact because pre-iPS cells can convert into iPS cells upon addition of ERK and GSK inhibitors.’

Finally, Austin Smith’s team noticed that their 2i process enabled the complete reprogramming of neural stem cells that intriguingly contained only very few copies of the ‘Yamanaka factor’ genes, supporting the suggestion that genetic manipulation of cells might not be obligatory for reprogramming them to pluripotency. The use of genetic reprogramming has been a key concern for those in the field, as put forward by distinguished British stem cell biologist, Fiona Watt and her postdoc Ryan Driskell who wrote: ‘ Discovering how the pluripotent state can be efficiently and stably induced and maintained by treating cells with pharmacologically active compounds rather than by genetic manipulation is an important goal.’

The paper by Smith and colleagues has gone on to be one of our research gems of the past 10 years and was picked out as a favourite by two of our Editorial Board members, Susan Gasser and Alfonso Martinez-Arias.

Looking back on this research, José Silva, who was a postdoctoral researcher at the time,  reminisces: “This work had a significant impact on my career, as it helped placing me on the path to becoming a Principal Investigator. Most importantly, it gave me a great platform to interrogate the underlying biology of nuclear reprogramming. “

See the Tenth Anniversary PLOS Biology Collection or read the Biologue blog posts highlighting the rest of our selected articles.

 

Silva J, Barrandon O, Nichols J, Kawaguchi J, Theunissen TW, & Smith A (2008). Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS biology, 6 (10) PMID: 18942890