Harvard team maps the interacting proteins behind stem cells' versatility.

Embryonic stem (ES) cells are plain enough to look at, forming a nondescript clump within the hollow ball of an early embryo. But that generic character is key to their magical rejuvenating potential. Unlike all other cells, which are preordained toward a specialized form and function, ES cells have a clean slate. The developing embryo can mold them into any cell type it needs—a trait called pluripotency.

		View Full Image This map sketches out the many proteins interactions that give embryonic stem cells their developmental prowess. Each circle represents a different protein, and each line indicates an interaction. The Orkin lab used the six proteins labeled in red as "bait" to fish for additional interacting proteins. The proteins that were "hooked" (green, blue, or yellow) were confirmed by further genetics experiments to be important for stem cell pluripotency. Reprinted by permission from Macmillan Publishers Ltd: Nature Vol 444/16 November 2006/doi:10.1038/nature05284, copyright 2006.

But what are the constituents behind that trait, and how do they work together? "It's been known for several years that there are a number of regulatory proteins required for pluripotency of ES cells," says Stuart H. Orkin, an HHMI investigator at Harvard Medical School. "But it's been unclear how these proteins relate to one another or whether they interact with other proteins."

To try to find out, Orkin and his team used a "guilt by association" approach built around a transcription factor called Nanog, previously shown to be an important regulator of pluripotency. By engineering mouse ES cells to express a custom-designed Nanog protein containing a sort of molecular "hook," the researchers anticipated that in retrieving that protein from cell extracts they might also pull along any other proteins with which it is associated. Those proteins, which the researchers could identify using mass spectrometry, were likely to participate with Nanog in controlling pluripotency.

The process worked as planned. Nanog that was retrieved from the cell extracts carried along 17 other proteins, several of which were transcription factors already known or implicated in mediating pluripotency.

To expand their search, the researchers outfitted several of the new proteins with the molecular hook and then repeated the experiment. They pulled new proteins from cell extracts, and also recaptured many of the same proteins that Nanog had captured, bolstering their hypothesis that these proteins operate in a common network.

But in science, as in courts of law, guilt by association isn't enough to get a conviction. So Orkin's team used genetic methods to deplete ES cells of many of their protein suspects. In most cases, the depleted cells began to differentiate into specialized forms, confirming that those proteins are required to maintain ES cells' developmental clean slate.

The researchers reported their findings—a protein interaction network map, or "interactome," of the key proteins regulating pluripotency—in the November 16, 2006, issue of Nature.

In all, the researchers drew an interaction network containing some three-dozen proteins—new information that could ultimately empower scientists to reprogram cells for medical therapies. "By knowing more about these proteins and their interactions with one another," Orkin suggests, we'll be poised to "differentiate ES cells into specific cell types in a directed manner."