:: By Michael Knezovich

:: Photography by Dan Dry

Investigations ::

Biologist Harinder Singh investigates how cells find their purpose in life.

In the bloodstream, every cell has a job. Neutrophils, the predominant white blood cells in pus, act as first responders, swarming toward cuts and scrapes or other tissue injuries to fight bacteria. Its life is purposeful but short, lasting only two or three days. Other white cells—macrophages—can live for years, patrolling the bloodstream like neighborhood beat cops, ingesting bacteria, debris from other cells, and other undesirables. Each white blood cell—six types in all—is genetically programmed for its job. Early in their development, though, macrophages and neutrophils are without identities, genetically capable of becoming either one. What mechanisms determine their fate?

Understanding the genetic mechanism that determines new cells' function, Singh says, could make it possible to create entirely new cell types for disease therapy.

Harinder Singh, Chicago’s Louis Block professor of molecular genetics and cell biology, has developed an approach that models the cell-fate mechanism as a circuit, much as an engineer designs an electrical circuit. But where an electrical circuit might map how a light is turned on or off, Singh’s circuit maps how genetic triggers turn on gene sets that determine whether a cell becomes a macrophage or neutrophil.

Described in an August 2006 Cell, Singh’s first cell-fate circuit holds broad promise, he says, that could help create entirely new cell types for therapeutic purposes.

The circuit—a mathematical model of the cell-fate mechanism—was developed using data from an elaborate experimental system. “In biology, generally speaking, people are experimentalists,” says Singh, also a Howard Hughes Medical Institute investigator. “We need to better integrate theoretical analysis with the traditional experimental analysis we’ve been doing in the past.”

Singh’s laboratory system flowed from efforts to identify the triggers that activate white-cell genes. Other researchers had already identified a protein called C/EBPa as a key genetic trigger for white-cell development. Singh had a hunch that another protein, PU-1, was a second white-cell trigger. To test the idea, his team genetically engineered a mouse lacking the ability to produce PU-1. When the animal then proved unable to generate white cells, it confirmed Singh’s hunch.

In nature, adult blood stem cells give rise to two cell types. One is simply another adult blood stem cell. The other, called a progenitor, has the capacity to become any kind of blood cell. Progenitors don’t stay progenitors for long, however, because genetic triggers send them along one or another developmental path, making experimentation impossible. “If you try to culture them,” Singh explains, “they either die or differentiate into the mature cells.” But in the engineered mouse, progenitor cells remained progenitors. “They’re stuck developmentally,” says Singh, “but they’ll happily divide in culture.” 

The customized cells gave the experimenters the power to turn on and off the genetic triggers and control their concentration in the cell. In one set of cells Singh’s team turned on a low level of the PU-1 protein. These cells weakly expressed the genes associated with both macrophages and neutrophils, as if the cells were previewing both possibilities before becoming one or the other. In another set of cells, the researchers turned on a higher level of protein. These cells initially exhibited the same weak, mixed-gene expression. But over time, the macrophage gene expression grew stronger, while the neutrophil gene expression disappeared altogether, and the cell became a macrophage.

The team also manipulated C/EBPa, the other white cell trigger. The results were similar, except for the outcome of the cell. When low levels of the C/EBPa were turned on, the cells exhibited the mixed-gene expression. When researchers turned on the protein at a higher level, neutrophil gene expression strengthened, macrophage gene expression faded, and the cell became a neutrophil. In both instances, when researchers turned up the volume on one trigger, it suppressed the other. As the PU-1 increased, C/EBPa decreased, and vice versa.Singh’s team had experimentally identified the circuit’s key inputs, or genetic triggers, and how they produced different outputs, namely macrophages or neutrophils. To create a corresponding mathematical model, Singh turned to Aaron Dinner, a physical chemist at Chicago.

Dinner charted the cell-development process as two differential equations, one accounting for cell development as a change over time, the other keyed to the activation and level of the genetic triggers. Programmed into a computer, the model mimicked the laboratory manipulations—increasing the trigger levels, for example. The computer-generated outcomes matched Singh’s experimental manipulations.

The macrophage/neutrophil model started in the laboratory and ended as a mathematical model. The ultimate goal, Singh says, is to reverse the process, so that computer models direct lab work. “We want the models to generate predictions that as biologists we cannot necessarily intuit. Then we would design experiments to test the model predictions.”

Such predictive models could provide new treatments for some leukemias. Because leukemia cells are, in effect, developmentally stuck, Singh believes that by tripping the cell’s circuits—introducing or increasing a genetic trigger, for example—they could be moved to a healthy state. Predictive modeling might also help researchers coax particular developmental paths from other types of adult stem cells, as well as from embryonic stem cells, which have the potential to grow into any cell type.

Singh envisions a step beyond the promise of stem cells. “If we understand circuitry well enough, we should be able to manipulate it to regulate large ensembles of genes. Imagine introducing new kinds of biomolecules into cells that confer on them a greater degree of motility or change their adhesive properties.” To expand his team’s expertise, he recruited a chemical engineer to a lab packed with biology postdocs. “If you think about it, engineers build circuits. They may be able to suggest approaches we have not been thinking about as biologists. They can say, ‘Here are the kinds of architectures I would use.’”

Singh continues: “Now, has nature used those sorts of architecture principles? We don’t know. But it’s a start.”