Researchers identify 'bridge' state necessary for cellular reprogramming

The discovery that fully mature cells can be reprogrammed to what are known as induced pluripotent stem cells by exposure to just a few proteins shocked the scientific world in 2006 and led to an explosion in stem cell research. But the exact molecular steps responsible for reprogramming the cells to become pluripotent — to have the potential to become any cell in the body — have remained murky.

Now researchers in the laboratory of Marius Wernig, MD, have shown that cells enter a previously unknown transition state identified by unique cell surface markers during their transition to pluripotency.

“This was completely unexpected,” said Wernig, associate professor of pathology at the School of Medicine and a member of the Stanford Institute for Stem Cell Biology and Regenerative Medicine. “It’s always been assumed that reprogramming is simply a matter of pushing mature cells backward along the developmental pathway. These cells would undergo two major changes: They’d turn off genes corresponding to their original identity, and begin to express pluripotency genes. Now we know there’s an intermediary state we’d never imagined before.”

The research was published in the April 1 issue of Nature. Wernig is the senior author of the research; graduate student Ernesto Lujan and postdoctoral scholar Eli Zunder, PhD, are the lead authors.

The researchers collaborated with others in the laboratory of Garry Nolan, PhD, professor of microbiology and immunology, to conduct the study. They used a technique developed in the Nolan lab called single-cell mass cytometry to analyze the expression of cell surface molecules on individual cells during the reprogramming process, and they grew individual cells separately in 96-well plates to ensure they were observing the orderly progression of events from just one ancestor cell.

Nolan, who is the Rachford and Carlota A. Harris Professor, and Wernig are also members of the Stanford Cancer Institute.

Achieving pluripotency

Pluripotent stem cells, by definition, can give rise to any cell in the body. Although embryonic stem cells are naturally pluripotent, induced pluripotent stem cells are created by scientists from existing adult cells, such as skin or blood. These iPS cells are easier than embryonic stem cells to come by and they match the genetic background of the person from whom they were obtained.

Lujan and Zunder found that, early in the reprogramming process, cells express proteins on their surfaces that are different from those found on either fibroblasts (the starting cell type) or fully reprogrammed iPS cells. Those proteins include CD73, CD 49d and CD200. When they looked more closely at this population of intermediate cells, they found that the cells were expressing genes for the transcriptional regulators Nr0b1 and Etv5 well before they began to express other genes such as Rex1, Dppa2, Sox2 and Nanog known to be involved in the acquisition of pluripotency.

“Previously we’ve assumed that cells undergoing reprogramming are simply retracing their developmental steps. Now we can say with high certainty that this is not the case,” said Wernig.

We’re learning more and more about how cells accomplish this really unbelievable task of reverting to pluripotency.

Much remains to be learned, however. It’s not clear, for example, what exactly Nr0b1 and Etv5 are doing in the cells early during reprogramming. “It’s clearly important, however,” said Wernig. “This program is absolutely necessary for the cells to achieve pluripotency.”

In addition to learning more about what happens in reprogramming, the researchers believe that isolating cells with intermediate-stage cell surface markers may help increase reprogramming efficiency for cell types that typically resist the transition to pluripotency.

“We’re learning more and more about how cells accomplish this really unbelievable task of reverting to pluripotency,” said Wernig. “Now we know that the cell biology of this process is novel, and this intermediary state is unique.”

Additional Stanford authors are graduate student Yi Han Ng and high school student Isabel Goronzy.

The research was supported by the California Institute for Regenerative Medicine, the National Science Foundation, the New York Stem Cell Foundation and Stanford’s Child Health Research Institute.

Stanford’s Institute for Stem Cell Biology and Regenerative Medicine and the Department of Pathology also supported the work.