With drug therapies for amyotrophic lateral sclerosis (ALS) showing minimal or no success, some researchers are considering whole-cell therapies. This week, two studies present proof-of-principle evidence that could place two new paving stones along the long and rocky road toward such treatments. In a Nature Neuroscience paper published online October 19, Nicholas Maragakis and colleagues at Johns Hopkins University in Baltimore, Maryland, show that astrocyte precursors, injected into a rat model for ALS, took hold in the spinal cord and yielded beneficial effects. In another study, published in Neurology October 21, Stanley Appel and colleagues at the Methodist Neurological Institute in Houston, Texas, attempted hematopoietic stem cell transplantation in human patients with ALS. While the patients received no benefit, the scientists were able to show that cells delivered intravenously reached the spinal cord.
Patients with ALS suffer motor neuron degeneration, leading to paralysis and ultimately death when the diaphragm can no longer support respiration. Recently, researchers showed that induced pluripotent stem cells can differentiate into neurons in vitro (Dimos et al., 2008 and see ARF related news story). However, studies have shown that glial cells, not just the neurons themselves, may be a primary mediator of disease (Yamanaka et al., 2008; Beers et al., 2006). The regular advances in stem cell research have led some to consider stem cell therapy for ALS and other neurodegenerative diseases, to replace if not the neurons themselves, then perhaps their support cells.
“When you’re transplanting a whole cell, there could be a multitude of things that these cells are doing,” said Maragakis, principal investigator for the Nature Neuroscience paper. ALS symptoms likely result from the confluence of multiple biochemical pathways. While a single drug can only affect a pathway or two, whole cells have the potential to exert multiple effects from one treatment. However, the complex nature of cells means that positive as well as negative effects are possible.
The Johns Hopkins team targeted astrocytes, which nourish motor neurons and recycle the neurotransmitter glutamate. First author Angelo Lepore and colleagues collected glial-restricted precursor cells (GRPs) from the developing spinal cord of donor animals, and injected these into immuno-suppressed rats expressing mutant human superoxide dismutase (SOD1), the gene associated with approximately 20 percent of familial ALS cases. Late-stage human patients (and mSOD1 animals) essentially suffocate when their diaphragm muscle fails; therefore, the investigators focused the injections on the C4, C5, and C6 vertebrae, where the nerves supporting respiration exit the spinal cord. The precursors survived and differentiated into astrocyte-like cells, and microgliosis was reduced. The treated rats survived an average of 17 days longer than control rats that received injections of dead GRPs or medium alone. (The average lifespan for the former was 173 days, versus 156 days for the latter.) Compound muscle action potential was also improved in the diaphragms of GRP-transplanted rats.
A primary job of astrocytes is to take up glutamate from the motor neuron synapse, via the glutamate transporter GLT1 (EAAT2 in humans). Glutamate transport is deficient in many ALS patients, and the accumulating glutamate poisons neurons (Maragakis et al., 2004). Indeed, riluzole, the only FDA-approved drug to slow ALS symptoms, acts on glutamate transport (Frizzo et al., 2004). Therefore, the researchers hypothesized that GLT1 could be responsible for the improvements in symptoms and lifespan.
When they analyzed GLT1 expression in the experimental animals, the scientists found that the rats carrying the SOD1 mutation had less GLT1 than wild-type rats, and GLT1 levels increased upon transplant of GRPs. When they transplanted GRPs from GLT1-null donors, however, GLT1 levels did not increase, suggesting the transplanted cells are primarily responsible for the increased GLT1 in recipients. GLT1 is unlikely to be the only thing that transplanted astrocytes provide, but clearly plays a central role. “There can be 20 things an astrocyte does, but this is a pretty big hit of what they do in a positive way,” said co-author Jeffrey Rothstein, also at Johns Hopkins.
Importantly, the transplanted cells did not replicate. They were partially differentiated, and could only turn into astrocytes or oligodendrocytes, depending on the cellular environment they landed in. “We want to be sure they don’t form tumors,” Maragakis said. “That would really be a disaster.” The experiments addressed this concern for the roughly two months that the rats in this disease model survived after the injection.
“It’s a good proof-of-concept paper showing how astrocytes can be protective,” said Appel, who was not involved in the Johns Hopkins study. However, he is skeptical of its ultimate clinical application, because the therapy might require multiple spinal cord injections. Astrocytes do not normally migrate across the blood-brain barrier. “We don’t want to save just a few neurons in local areas,” he said.
Ultimately, Maragakis envisions a one-time therapy in which patients receive cells through spinal fluid injection. He is working with biopharmaceutical company Q Therapeutics, based in Salt Lake City, Utah, on further preclinical experiments on cell therapies for ALS. “We have animals now that are carrying these cells,” Maragakis said. It is not yet clear if a one-shot spinal cord treatment would be possible. And because a rat or other animal model is not a human, it remains to be shown that the therapy is safe and effective for people.
For his own transplant study, Appel bypassed animals and went straight to human patients. ALS is accompanied by inflammation, and Appel hypothesized that stem cell transplants, which proved beneficial in other neuroinflammatory diseases (Wada et al., 2000; Biffi et al., 2004; Biffi et al., 2006), might help ALS patients. While Appel did not expect stem cells to replace dying neurons, he hoped to repair those neurons. Maragakis called Appel’s experiment “a very bold thing to try.”
Appel and his colleagues treated six people with sporadic ALS by infusing donor hematopoietic stem cells (HSCs). The cells came from siblings with matching human leukocyte antigens (HLA). The donor cells fully replaced the recipients’ blood cells in four of the patients, who nonetheless derived no clinical benefit.
However, Appel is the type to make lemonade when given lemons, and he was able to extract useful information from the experiment. “If we had not done the human study, we would not have learned a tremendous amount,” he said. Most importantly, the paper confirms that donor cells traversed the blood-brain barrier. It had previously been shown that the occasional transplanted stem cell made its way to the brain (Mezey et al., 2003), but Appel’s study was the first to find plentiful donor cells in spinal cord sections. Intravenous therapy has the potential to be more comfortable and convenient for patients, and at least in these six volunteers, this treatment appeared safe. Although HSCs alone did not help his patients, Appel suggests the cells could serve as a “Trojan horse” to deliver gene therapy.
“These cells could conceivably be very useful if you could figure out a way to make them carry and release protective substances into the damaged area,” wrote Barbara Crain of Johns Hopkins, who was not involved in either study, in an e-mail to ARF. The “Trojan horse” approach has already been used to deliver stem cells that produce a neurotrophic factor to muscles, increasing survival of a rat ALS model by nearly a month (Suzuki et al., 2008).
Stem cell transplantation holds potential to treat not only ALS, but other neurodegenerative diseases, as well. For example, an October 1 paper in the Journal of Clinical Investigation examined neural stem cell transplants for an animal model of spinal muscular atrophy (SMA), a common genetic cause of infant mortality characterized by muscle weakness. In a mouse model of SMA, first author Stefania Corti, principal investigator Giacomo Comi, and colleagues at the University of Milan in Italy showed that transplanted neural stem cells generated motor neurons, alleviated symptoms, and extended lifespan.
However, any cell therapy is, at minimum, years away. “There are many hurdles to get to that point,” Rothstein said. As with any new treatment, someone would have to work out dosage, specificity, delivery method, and quality control. This is normally the job of pharmaceutical companies, which are experienced in drug development but not in cell therapies. “It’s an industry that’s absolutely in its infancy in how to do these things,” he said. And, he noted, cell therapy is decades behind gene therapy, which has yet to reach a large number of patients.
Stem cells are promising, but “not ready for prime time,” Appel agreed. The current experiments inch a bit further along the road to viable ALS cell therapies, but the destination remains far off.—Amber Dance
Amber Dance is a freelance writer in Los Angeles.
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