By rewinding the clock and coaxing mature muscle back to an earlier stem cell stage, bioengineers from the University of California (UC), Berkeley, in the US have opened the door to the development of new ways to treat muscle degeneration such as that seen in muscular dystrophy or aging. They also accomplished the task by altering cell chemistry without resorting to gene manipulation.

They write about their work in the 23 September issue of the journal Chemistry & Biology.

Skeletal muscle tissue is made of myofibers, long, fused bundles of individual muscle cells (myoblasts). The fusion of these individual cells is the last stage of differentiation of skeletal muscle, which started as undifferentiated stem cells.

Using mice, the researchers not only found they could coax the mature muscle tissue back to an earlier stem cell stage, but they found the newly reprogrammed stem cells could also be used to repair damaged tissue.

Principal investigator Irina Conboy, UC Berkeley assistant professor of bioengineering, told the media they used tiny chemicals called molecular inhibitors to de-differentiate mature tissue, sending fused myofibers back to an earlier stage, an approach that is increasingly sought after in the stem cell field.

“These tiny chemicals go inside the cell and change the way the cell behaves without changing its genome,” said Conboy, who is also a member of the Berkeley Stem Cell Center and an investigator with the California Institute for Quantitative Biosciences (QB3).

“The inhibitors were only used for 48 hours, enough time for the fused myofibers to split into individual cells, and then they were washed away. The cells can proceed to live and die as normal, so there is no risk of them dividing uncontrollably to become tumors,” she added.

For a long time scientists thought muscle formation was a one-way street: once the journey from stem cells to myoblasts to muscle fiber was complete, there was no going back. But Conboy said “we were able to get a multi-nucleated muscle fiber to reverse course and separate into individual myoblasts”.

There are a number of approaches in stem cell research, and most treatments rely on pluripotent stem cells, the type that can turn into any type of cell. These can come either from embryonic tissue, a controversial method because it uses embryos, or from adult, differentiated cells that have been reprogrammed through gene insertion to de-differentiate into embryonic-like state, into what is called induced pluripotent stem cells (iPS).

Pluripotent stem cells can go on dividing and proliferating indefinitely, a problem in itself, because if they are not driven toward a particular type of organ, they can quickly become teratomas, types of tumor that just contain a mass of malformed tissue.

Lead author Preeti Paliwal, a post-doctoral researcher in bioengineering at UC Berkeley, said:

“The biggest challenge with both embryonic stem cells or iPS cells is that even a single undifferentiated pluripotent cell can multiply in vivo and give rise to tumors.”

But in this study the researchers did not turn the clock back as far as pluripotent cells: they only went as far as the progenitor cell stage. Progenitor cells are already destined to become a particular type of tissue: muscle progenitor cells can only form muscle tissue and liver progenitor cells can only form liver tissue.

Paliwal pointed out that when transplanted into live muscle tissue, the progenitor cells do not go on to form tumors.

“… we focused on the progenitor cell stage, in which cells are already committed to forming skeletal muscle and can both divide and grow in culture. Progenitor cells also differentiate into muscle fibers in vitro and in vivo when injected into injured leg muscle,” said Paliwal.

To perfect the method, the researchers had to overcome a number of problems, including the one of cell apoptosis. When confronted with signals to start dividing again, such as the inhibitors used in this study, cells can become confused and start commiting suicide, or cell apoptosis.

So to overcome this, the researchers also used an apoptosis inhibitor:

“We basically brainwashed the cells to go into the cell cycle, to divide and also not die in the process,” said Paliwal.

The researchers now want to test their method on human muscle tissue and screen for other molecules that could help de-differentiate mature tissue.

Conboy said they don’t think this method will work for all degenerative diseases: it might work for some, where you can start with differentiated tissue like brain cells or liver cells:

“But patients with type I diabetes, for instance, lack the pancreatic beta-islet cells to produce insulin, so there is no functional differentiated tissue to start with,” said Conboy.

“Our approach is not a replacement for pluripotent cells, but it’s an additional tool in the arsenal of stem cell therapies,” she added.

Funds from the The National Institutes of Health and the California Institute of Regenerative Medicine helped pay for the study.

Note: this article later amended to correctly state embryonic tissue comes from embryos, not fetuses. Our apologies if this incovenienced our readers.

Written by Catharine Paddock PhD