Part 2 of a 3-part series. See also part 1 and part 3, download .pdf of series.
5 October 2010. From fibroblasts, to stem cells, to neurons—it is a vision that holds the promise of someday treating amyotrophic lateral sclerosis (ALS), or at the least helping scientists to better understand the disease. At the Fondation André-Delambre’s annual symposium, held September 24-25 in Québec City, Canada, several researchers reported progress toward that still-distant goal. Researchers are using embryonic stem cells as well as induced pluripotent stem (iPS) cells, (see ARF related series) to make motor neurons, which they then study in culture. Some are using these artificially made motor neurons to examine the key question of which cell types truly cause ALS. Others are applying stem cell tools to understand why motor neurons in particular are susceptible to degeneration. Another question is which kinds of motor neurons are in greatest danger. Victor Rafuse of Dalhousie University in Halifax, Canada, presented data on susceptible motor neurons that fatigue quickly versus resistant motor neurons that fatigue slowly. His results to date suggest, to scientists’ dismay, that when they manufacture motor neurons from stem cells, the resulting neurons are most similar to the natural fatigue-resistant motor neurons—the ones least likely to degenerate in ALS.
Among the top news: Kevin Eggan of Harvard University announced that his long-awaited patient-specific iPS cell lines are available for the asking. He has both SOD1 and TDP-43 mutant varieties. “Much exciting research using this new model is expected in the future,” wrote meeting attendee Christine Vande Velde of the Université de Montréal in Canada, in an e-mail to ARF. [For a comprehensive summary on iPS cells in neurodegeneration, see Madolyn Bowman’s series on Alzforum.]
A central question in ALS research has been which cells actually inflict the damage. Motor neurons are the ones that die, but studies in some model systems suggest that glia instigate the disease in a non-cell autonomous fashion (see ARF News story on Nagai et al., 2007 and Di Giorgio et al., 2007; and ARF News story on Clement et al., 2003). One treatment possibility, then, would be to replace endogenous, damaging microglia with new ones that do not carry mutations. Nicholas Maragakis of the Johns Hopkins School of Medicine in Baltimore, Maryland, is working on transplanting glial-restricted precursor cells (GRPs) into rodents, where they differentiate into astroglia. In Québec, Maragakis reported that he is having some trouble getting human GRPs to differentiate in the same way, and to spread beyond the injection site. He suggested that in the meantime, the transplantation of GRPs into animals of different genotypes could be useful in the lab to understand how different cell types interact in the disease.
Much of the work Maragakis discussed was performed by Angelo Lepore, who derived GRPs from fibroblasts of mice harboring an ALS-linked mutation in superoxide dismutase 1 (SOD1), and injected them into the brains of healthy rats. This is a ‘mechanical’ counterpart to chimeras other researchers have created genetically using the Cre/Lox system to selectively excise or express mutant SOD1 (mSOD1) gene in different cell types. The injection protocol has the advantage that it transplants restricted cell lineages locally, Maragakis said. The majority of GRPs develop into astrocytes. In addition, he noted that scientists can do “mix-and-match biology,” putting wild-type cells into one brain hemisphere and mutant ones into the other, for built-in controls. The disadvantage, he said, is that the injected cell type is immature and will not necessarily integrate with the native neural network.
So far, the researchers have observed the transplanted animals but for three months. In this time, they noticed ubiquinated inclusions, loss of motor neurons, and weakened grip in animals that receive mSOD1 GRPs. “This is not recapitulating ALS,” Maragakis said. “I think of it rather as influencing motor neuron vulnerability.”
For his part, Eggan presented preliminary work that may help scientists understand the relative contributions of motor neurons and astrocytes to ALS. Evangelos Kiskinis and Sophie DeBoer in the laboratory are working with long-term, time-lapse microscopy to examine how stem cell-derived motor neurons survive in the presence of wild-type or mSOD1 astrocytes. When the scientists plated motor neurons on wild-type mouse astrocytes, the neurons put out a dense network of processes over the course of two weeks. When plated on astrocytes from mSOD1 mice, the motor neurons initially behaved similarly. But after about a week, the neurons retracted their processes and died. “The cells never [achieve] the same complexity of processes that you see in the control experiment,” Eggan said. “It really does seem like we are [mimicking] some sort of effect—which is going on very early in the lives of these [ALS model mice]—in tissue culture.”
Yet another central question in ALS research is why the motor neurons, of all cells, are the ones that perish. Christopher Henderson of Columbia University in New York City is interested in how motor neurons respond to environmental toxins such as organophosphates. Used in solvents, plasticizers, lubricants, and fertilizers, organophosphates have come up as a possible explanation for high ALS rates among farm workers and Gulf War veterans (Horner et al., 2003). Using neurons derived from embryonic stem cells, he and colleague Marine Prissette found that motor neurons are more likely to die from organophosphate treatment than other types of neurons.
ES- or iPS-derived motor neurons have many advantages, including the ability to create patient-specific lines that carry human mutations associated with ALS (see ARF News story on Dimos et al., 2008), but it is not clear if cultured cells are really similar to the motor neurons that die in ALS. In fact, that might not be the case, Rafuse said. Motor neurons, he noted, are heterogeneous. Some fatigue quickly—such as the small motor neurons innervating the arm muscles we work at the gym—and others are fatigue-resistant—for example, those controlling the neck muscles that hold up our heads all day long. Fatigue-resistant motor neurons are less susceptible to ALS (see ARF News story on Saxena et al., 2009).
The standard trick researchers use to convert stem cells into motor neurons is to add the developmental regulators Sonic hedgehog and retinoic acid. Rafuse found that most motor neurons coming out of these protocols express the fatigue-resistant neuron marker Lhx3, not the fast-fatiguing marker Lim1 (Soundararajan et al., 2006). And the work of Sam Pfaff at the Salk Institute in La Jolla, California, suggests that when injected into animals, the stem cell-derived motor neurons grow toward fatigue-resistant postural muscles. What’s more, they even convert some fast-fatiguable muscle into fatigue-resistant muscle.
In other words, the stem cell-derived motor neurons scientists are studying may be those that are least susceptible to degeneration in ALS. “This work has important implications for the field, and we will need to keep this in mind as we re-interpret earlier works using these cultured neurons,” Vande Velde wrote.
“Excitingly,” Vande Velde added, “he has identified a ‘recipe’ to make large motor neurons, which are the ones lost in ALS.” Rafuse found that conditioned media from neural progenitors, in addition to Sonic hedgehog and retinoic acid, pushes stem cells toward the fast-fatiguing phenotype. Cells from these cultures express Lim1 and, when transplanted, grow toward fast-fatiguing muscles in the limbs. The work suggests that scientists will be able eventually to develop protocols for turning stem cells into motor neurons that are susceptible to ALS pathology.—Amber Dance.
See also part 1 and part 3, download .pdf of series.