Scientists knew that the DNA of a cell would need to be reprogrammed to turn it into an iPS cell. They also knew that the four genes they added would be predicted to do just that. What they didn't know was what the specifics of the reprogramming were.
Every cell has the same DNA. And in that DNA is the same set of genes. What makes one cell different from another is what genes are on and what genes are off.
So when a cell develops into a new kind of cell, its DNA undergoes changes and is reprogrammed. New genes get turned on and old genes get shut off. There are a couple of ways this can happen.
One is by using special genes called transcription factor
(TF) genes. These genes have the instructions for making a kind of protein called a transcription factor (TF). A TF's job is to turn genes on and off.
The four genes the researchers add to make an iPS cell are all TF genes. When the researchers looked at the genes of a fibroblast and the genes of a successful iPS cell, what they saw was lots of different genes on and off.
Genes involved in being an ES cell were now turned on. And genes that were involved in being a fibroblast were turned off. So the TFs were having global effects on what genes were turned on and off.
Another way that genes are turned on and off is by something called DNA methylation
. This basically is just a way to shut a gene off by adding a chemical group to the DNA near a gene. If a gene has lots of methyl groups, it is off. And if it has few or no methyl groups, it is on.
Scientists knew that once a cell stopped being an ES cell, the ES genes became heavily methylated and were shut off. As expected, ES genes lost the methylation when a fibroblast became an iPS cell.
So now these researchers had a pretty good feel for what the successful iPS cell looked like. The next step was to look at unsuccessful ones to see what went wrong. The hope was that by learning this, they'd be able to push more cells into becoming iPS cells.
Too Much Methylation
Scientists can increase the
efficiency of making iPS cells
by decreasing DNA methylation.
The researchers looked at three failed cells. These cells made it some of the way towards being an iPS cell but stalled out on the way. When these cells are allowed to continue to grow and divide, a few do go on to become iPS cells but most stay in the stalled state.
When the researchers looked closely at these cells, they saw that they failed for different reasons. But one thing they had in common was too much methylation at important ES cell genes. So the researchers reasoned that if they could get rid of some of the methylation, then these cells might turn into iPS cells. And that's just what happened.
There is a chemical called AZA that keeps DNA from being methylated. The researchers added this chemical to the stalled cells for 48 hours. The results were impressive.
Let's look at one cell, MCV8, as an example. After five generations without AZA, around 0.41% of MCV8 cells went on to become iPS cells. Not very efficient.
When AZA was added for 48 hours, 77.8% of the MCV8 cells went on to become iPS cells. This is obviously a huge difference. But can AZA help if added at the beginning of the process before cells stall? Yes, if it is added at the right time.
The scientists started the process of turning a fibroblast into an iPS cell. Then they added AZA 4, 6, or 8 days later.
The cells that got AZA at 4 or 6 days all mostly died off. But when added at 8 days, AZA resulted in four times as many iPS cells being created.
The researchers don't know yet exactly which genes AZA targets in these cells. Once they figure this out, they may be able to come up with even better ways to create more iPS cells that might one day be able to treat and possibly cure untreatable and incurable illnesses.