Stem cells are hailed for their ability to develop into any type of cell, often stepping in to replace tissue lost to disease or injury. But these versatile youngsters have still more potential: They can act as mini-laboratories, allowing scientists to unravel the course of a disease and test possible therapies. Two teams of researchers have used this approach to study one of the most elusive neurological disorders: amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig’s disease.
Fred H. Gage, Carol Marchetto and co-workers at the Salk Institute in La Jolla, Calif., and Kevin Eggan and colleagues at Harvard University used embryonic stem cells to generate a supply of human motor neurons, the type of cell destroyed in ALS. The work provides clues about the importance of astrocytes—“support” cells for neurons—in ALS, opening up potential new treatment approaches.
ALS has yielded few clues to explain its devastating course, which begins with muscle weakness and usually leads to paralysis and death. A small number of patients show mutations in a gene for the enzyme superoxide dismutase 1 (SOD1), which helps protect cells from the destructive molecules known as free radicals. (These unstable compounds produce oxidative damage, “rusting” the cells they encounter, and ALS sufferers show evidence of oxidative damage.)
Scientists have used SOD1 mutations to develop lines of transgenic mice and rats with ALS-like disorders. But several treatments that have shown promise in these rodents were failures in human clinical trials. To develop better drugs, researchers needed human motor neurons, but according to Gage, it isn’t practical to obtain mature motor neurons from human donors. “Even assuming you could remove the neurons intact, they wouldn’t survive in culture,” he says. Scientists were at an impasse.
Some researchers, though, have long believed that stem cells could be used to produce a desired cell type in sufficient quantities. In 2002 a team led by Tom Jessell at Columbia University finally guided mouse embryonic stem cells to develop into motor neurons.
In 2006, meanwhile, Donald Cleveland and colleagues at the University of California, San Diego, provided a vital clue into the development of ALS. The death of motor neurons was not exclusively due to a problem within the neuron itself. Nearby astrocytes—which do not transmit signals but carry out other important functions—also contributed to the disease.
These two lines of research converged in 2007 when two studies, including one from Eggan’s laboratory, involved using motor neurons derived from mouse embryonic stem cells to confirm Cleveland’s findings. The researchers demonstrated that motor neurons carrying mutated SOD1 showed abnormalities but survived when alone. The mutated gene led to neuron death only when it appeared in nearby astrocytes. In fact, even perfectly healthy motor neurons died when cultured with astrocytes carrying the SOD1 mutation. The healthy neurons did survive longer, however, suggesting that in mutated motor neurons, some intrinsic problem still plays a role in the cell’s demise.
Next, researchers asked whether the same principle held true in human ALS. In the December 4, 2008, issue of Cell Stem Cell, Gage and co-workers used human embryonic stem cells to generate a supply of motor neurons. The group then cultured the neurons along with astrocytes (also derived from stem cells), some normal and some carrying a mutated form of SOD1.
The investigators found that only half as many motor neurons survived when exposed to the mutated astrocytes, compared with those cultured with the normal version of the gene. They also found evidence that the astrocytes were producing superoxide (a damaging form of oxygen), which activated a gene called NOX2 in the neurons.
When motor neurons were exposed with the antioxidant apocynin (a NOX blocker), the neuronal survival nearly doubled, even in the presence of astrocytes with the mutated gene. In the astrocytes, apocynin treatment cut superoxide production by almost half.
“Our data support the notion that in ALS, astrocytes with the SOD1 mutation can activate NOX2 to produce oxygen radicals, and treatment with antioxidants can reverse this process,” Gage says.
He cautions, however, that apocynin may be too toxic for use in humans. “We are using our stem-cell screening method,” he says, “to come up with drugs that have a better safety profile.”
In the same issue of Cell Stem Cell, Eggan and colleagues used embryonic stem cells to develop a line of motor neurons with a different mutation of SOD1. This study, too, confirmed that mutated astrocytes are toxic to motor neurons. The study found that the mutation caused another gene to be overactivated: PGD2, which codes for a receptor for one of the prostaglandins, a biologically important family of lipids. That led researchers to expose motor neurons with PGD2, since prostaglandins play a role in inflammation, a hallmark of ALS.
The results were striking: Motor neurons exposed to PGD2 showed a dramatic decrease in number after 20 days, while a compound that blocks this receptor led to a 32 percent increase in survival.
The research points to PGD2 and NOX2 as targets for potential new ALS medications. The technique may also help explain why some compounds that performed well in mice—including a drug that targets one of the prostaglandins—did not pan out in clinical trials. Eggan and colleagues, for example, reported that the toxic effect of the SOD1 mutation on human motor neurons was even stronger than had been observed in mouse motor neurons. Having an abundant source of human motor neurons should help researchers clarify the difference.
But as a more general, more immediate benefit, the work establishes that a stem-cell-based screening method is both possible and important.
Eva Hedlund of the Ludwig Institute for Cancer Research Ltd. and the Karolinska Institute in Stockholm, who co-authored an accompanying commentary, says that since the cause of most ALS cases remains a mystery, studies that trace the disease’s progression and identify therapeutic candidates are important.
She notes that 90 percent of ALS patients have the “sporadic” form of the disease, which does not run in families and can’t be traced to any one gene. “There is currently no way to diagnose sporadic ALS before symptoms begin,” she says. “Therefore, substances that can extend the lives of already affected individuals represent a higher priority for clinical development.” She adds that the stem-cell-based systems could help scientists further investigate the disease’s onset.
Using induced pluripotent stem cells (iPS cells)—cells taken from the patient’s own skin and reprogrammed into stem cells, then coerced into developing into motor neurons—is a long-term goal. A team led by Eggan took a first step in a study published in the August 28, 2008, issue of Science. The investigators used fibroblasts, or skin cells, from an 82-year-old woman with ALS to generate a supply of stem cells that developed into motor neurons.
“It is particularly encouraging that neither the advanced age nor the severely disabling disease of this patient prevented us from reprogramming her fibroblasts,” the authors wrote. They added that especially in the case of sporadic ALS, iPS cells would carry “the precise constellation of genetic information” associated with that person’s condition, one day leading to individualized treatment.