Neurosurgeon Ivar Mendez of the University of Saskatchewan often shows a video clip to demonstrate his work treating Parkinson's disease. It features a middle-aged man with this caption: “Off medications.” The man's face has the dull stare typical of Parkinson's. Asked to lift each hand and open and close his fingers, he barely manages. He tries but fails to get up from a chair without using his hands. When he walks, it is with the slow, shuffling gait that is another hallmark of Parkinson's, a progressive neurological disorder that afflicts an estimated one million Americans, most of them older than 60.
Then the video jumps forward in time. The same man appears, still off medications. It is now eight years since Mendez transplanted dopamine cells from a fetus into the patient's brain. These neurons, which live in a midbrain region called the substantia nigra and secrete the neurotransmitter dopamine to initiate movement, are the ones that die off in Parkinson's. The man has aged, but his energy and demeanor are characteristic of a much younger man. Asked to do the same tasks, he smoothly raises his arms high and flicks his fingers open and shut rapidly. Arms crossed on his chest, he rises from a chair with apparent ease. Then he struts down the hall.
In the 25 years since the first few patients received transplants as part of a clinical trial at University Hospital in Lund, Sweden, hopes of using cell-based therapy as a treatment for Parkinson's have repeatedly risen and then been dashed. Stem cells are a biological raw material of enormous potential because they can generate new cells through the ability to divide indefinitely and to give rise to specialized cells. These cells can then be used to repair brain damage from degenerative disorders such as Parkinson's. Stem cells, however, have been hard to come by. So far the cells transplanted in humans have been derived from aborted fetal tissue, although scientists have also transplanted stem cells derived from human embryos and adult skin cells into animals. Thorny political and ethical issues limit access to both fetal cells and embryonic stem cells, and fetal cells are in particularly short supply. Two large clinical trials using fetal tissue, published in 2001 and 2003, were considered failures because of their widely variable results; not enough patients improved by the study end points, and some developed serious side effects. Many scientists gave up on cell therapy.
But a handful of laboratories persevered. Now new evidence showing that transplantation can work well, as in Mendez's patient, and possible new sources of cells free of ethical concerns have sparked a fresh optimism. This year neurologist Roger A. Barker of the University of Cambridge plans to lead the first large clinical trial of cell therapy for Parkinson's in a decade. “We've broken through the old barriers,” says cell biologist Ole Isacson of Harvard University.
The momentum most likely will propel cell therapies for other disorders as well. Researchers are trying to apply the technique to more than a dozen diseases, including diabetes, spinal cord injury and several forms of cancer [see “Stem Cell Repair Shop,” on page 81]. In addition to Parkinson's, the most significant progress has been made with retinal diseases. Clinical trials are under way to use retinal pigment epithelial cells for treatment of macular degeneration. According to the California Institute for Regenerative Medicine, theoretically there is no disease to which stem cell therapy could not be applied. In each case, the requirements depend on the difficulties inherent in generating the specific type of cell scientists hope to replace.
Progress in Parkinson's has been particularly promising, Isacson says, because “it's easier to solve.” The debilitating movement difficulties characteristic of the disease have a relatively straightforward cause: dopamine loss. And researchers were able to generate dopamine neurons from stem cells quite quickly. Cell therapy typically leads to restored mobility and function—improving patients' gait, for instance, and reducing tremor—but does not ameliorate every aspect of Parkinson's. Patients may still suffer from dementia, gastrointestinal problems and sleep disorders, for instance. Yet in the best-case scenario, patients could gain 20 to 30 years of excellent quality of life with a single intervention and require virtually no medications. “You've not cured the disease,” Barker says, “but you've transformed the natural history of Parkinson's disease.”
A mild tremor in the hand or some other extremity is often the first sign of Parkinson's. Tremors are followed by rigidity in the muscles, a stooped posture and the distinctive difficulty walking first described by James Parkinson in 1817. The movement difficulties relate to the loss of a dopamine neuron called A9 in the substantia nigra, which among other things controls the initiation of motion. By the time the first tremor appears, patients have already lost about 70 percent of those A9 neurons—a threshold that is like a water level, Isacson says. They hit the water and begin to sink under a flood of movement troubles.
Since the 1960s Parkinson's has been treated with medications that replace missing dopamine in the brain. L-Dopa is a dopamine precursor, and doses of this small molecule cross the blood-brain barrier and enter brain cells, which convert L-Dopa into dopamine and release it. Other drugs, known as dopamine agonists, stimulate dopamine receptors in the absence of the neurotransmitter, thereby mimicking its effects. The medications improve parkinsonian symptoms, but their benefits diminish over time, and they carry side effects such as alternating periods of mobility and immobility and the emergence of additional jerky movements.
In the 1990s clinicians developed an alternative therapy called deep-brain stimulation (DBS), the surgical insertion of an electrode that delivers electrical pulses to directly alter neuronal activity in a specific area of the brain. The treatment can work well. At the University of California, San Francisco, Medical Center, for example, 45 to 70 percent of patients who receive DBS for Parkinson's improve. Yet over time, patients begin to decline again because the electrode stimulation can no longer compensate for the continuing loss of dopamine. Cell-based therapy, in contrast, is designed to directly restore the cells lost in the disease process.
The earlier large clinical trials of cell therapy suffered from multiple problems. For example, it now appears that some of the patients selected were too old and their disease too advanced to get good results. Instead of infusing a substance containing a single type of cell, surgeons transplanted chunks of tissue, which included other material that triggered immune reactions. The procedure itself was conducted differently by every team. Moreover, the end points for the studies were too short—neither was more than two years—for the transplanted cells to take full effect.
Of the patients who have received cell-based therapy for Parkinson's, those transplanted by Mendez's team have done best. Mendez began transplanting fetal cells into patients in the late 1990s, when he was at Dalhousie University in Nova Scotia. He improved the preparation of the cells by treating them to encourage growth and creating pure cell suspensions instead of transplanting chunks of tissue. Using a computerized injector that he developed to standardize the process, Mendez targeted two brain areas instead of one—the substantia nigra, where dopamine cells naturally originate, and the putamen, which their axons need to reach. All 10 of his patients improved significantly on the standard Parkinson's rating scale, which measures the course of the disease. In a separate postmortem analysis of five patients published in 2008, Mendez and Isacson, who have been collaborating for about 10 years, found that the grafted neurons survived without signs of degeneration for as long as 14 years. “Methods matter,” Mendez says. “We now have all the experience and the techniques and the instruments that will be able to plant these cells safely into the human brain.”
New Kinds of Cells
The biggest remaining challenge is obtaining enough viable stem cells. The fetal cells implanted to date have been harvested from the midbrain of an aborted fetus aged six to nine weeks. Such stem cells have already differentiated into dopamine neurons yet retain the capacity to generate more new neurons after transplantation. Still, fetal cells “are not the answer,” Mendez acknowledges. Politics aside, there will never be enough for all the patients who would need them.
Another possibility emerged in 1998, when cell biologist James A. Thomson of the University of Wisconsin–Madison and his colleagues derived the first embryonic stem cell line. They were working with the blastocyst of a human embryo, a brief early developmental stage when the ball of cells contains an inner clump of 20 to 30 cells that are capable of growing into any of the more than 200 types of adult cells in the body. Unlike fetal tissue cells that have started down the path to differentiation, these so-called pluripotent stem cells have the potential to produce any type of tissue in the body.
Thomson's team removed those cells and nurtured them in the lab so that they divided. The result was an infinitely renewable lab-maintained source of stem cells—a cell line—that would not require further new embryos. The ethics were still complicated by the original use of embryos, but suddenly large-scale cell-based therapy seemed achievable. The challenge was to coax those embryonic stem cells to develop into the specific cells needed to treat a disease—dopamine neurons for Parkinson's, for instance, or insulin-producing cells for diabetes.
Also in 1998 Isacson's group reported that it had done just that in mice. The researchers differentiated A9 neurons from mouse blastocysts. When they injected those A9 cells into a mouse brain, they found that the cells lived and formed connections with the other neurons in the brain. In 2002 Isacson's group showed that the same procedure restored movement and mobility in a rat with a drug-induced version of Parkinson's. Several other groups achieved similar recovery in rodents. Immediately researchers tried to create A9 neurons from human embryonic stem cells—but that step proved more difficult. “For nearly 10 years that was largely a failure,” says cell biologist Lorenz Studer of the Memorial Sloan Kettering Cancer Center. “We would have expected the cells to behave well, but they did not.”
A breakthrough with an alternative approach came in 2007, when the team of biologist Shinya Yamanaka of Kyoto University in Japan figured out how to create stem cells from an adult's own tissues. Beginning with adult mouse skin cells, Yamanaka's team “reprogrammed” the cells biochemically, driving them back to something resembling an embryonic stem cell, which could then be used as a basis for deriving a totally different kind of body cell, such as a neuron.
In essence, Yamanaka's group had found a way to create a limitless supply of stem cells from adult skin cells, thereby sidestepping the political and ethical issues that surround research with embryos. The accomplishment won Yamanaka the 2012 Nobel Prize in Physiology or Medicine. Furthermore, if the cells, which are called induced pluripotent stem cells, always originate with the individual patient being treated, the considerable risk of immune rejection would disappear. “They solved a very big problem,” says Mahendra Rao, former director of the National Institutes of Health's Center for Regenerative Medicine and now vice president of regenerative medicine at the New York Stem Cell Foundation.
Newly Nimble Monkeys
A year after Yamanaka's discovery, Isacson's team showed that it could create A9 dopamine neurons from such reprogrammed adult rodent cells. The scientists soon began putting the new cells in mice and rats with signs of Parkinson's, and in 2008 they reported improved function. Then they turned to nonhuman primates. Working with a monkey with drug-induced Parkinson's, Isacson's group harvested the monkey's skin cells, drove them back to an embryonic state, then differentiated them into dopamine neurons and put them into the monkey's brain. For two years, they monitored the monkey. In results presented at conferences late in 2013, they showed that according to positron-emission tomography (PET) scans the grafted dopamine neurons had survived and grown. About eight months after the transplant, the monkey's motor disorder ceased. A postmortem analysis showed that the new neurons had made connections with other neurons throughout the brain area where they had been grafted.
The same year two other groups also reported success with adult-derived stem cells and monkeys, including the lab of cell biologist Su-Chun Zhang of the University of Wisconsin–Madison and Yamanaka and his colleague Jun Takahashi. “All three groups now demonstrate pretty unequivocally that the graft can survive, can differentiate into the right type of cells and then can integrate into the brain structurally,” Zhang says.
Isacson's monkey is the only one to be observed for a longer period—two years—and to have shown functional recovery. The researchers are pursuing longer-term studies with more monkeys to convincingly show both safety and efficacy. Clinical trials could follow, possibly within a few years, say Mendez and Isacson, who are convinced that these adult-derived cells are the future.
Others are still betting on embryonic stem cells. In 2011 and 2012 Studer's lab and that of neurobiologist Malin Parmar of Lund University successfully differentiated human embryonic stem cells into dopamine neurons. When grafted into a mouse, rat or monkey with parkinsonian symptoms, these cells survive and lead to recovery of function. Late in 2014 Parmar reported the creation of even better A9 neurons. More stringently tested, they function as well as fetal cells, growing axons over equally long distances, targeting the correct areas and restoring motor function in rats. “Our hope is that they are ready for clinical trials in about three years,” Parmar says.
Studer, too, is optimistic. He recently received a $15-million grant to perfect his technique and generate cell lines based on GMP (good manufacturing practice) guidelines. “Now we have a protocol that led us to say we might actually be ready,” Studer says. In parallel with the work manufacturing large batches of cells, he plans to begin lining up patients for a clinical trial, most likely the first to use embryonic stem cells.
Scientists at International Stem Cell in Carlsbad, Calif., have taken a different approach. Late in 2014 they reported success creating safe and effective dopamine neurons from unfertilized eggs that are chemically induced to develop as if they had been fertilized, a process called parthenogenesis that avoids the use of embryos. The company is seeking regulatory approval to begin a clinical trial with its human parthenogenetic neural stem cells early in 2015.
One reason for sticking with embryonic stem cells is regulatory. “This is all new ground,” says University of Cambridge's Barker. “Cells are not a drug, and they are not a device. What are they?” To date, stem cells have been regulated by line—a set of renewable cells that are cultured in one lab and deemed safe. If stem cells are produced for individual patients—using the full potential of the newest technology—and still are required to follow the same approval process as existing stem cell lines, the therapy would be cost-prohibitive. One solution is to approve a generic induced stem cell process rather than separate lines. Another answer, which sacrifices some immune response benefits, would be to create a bank of up to as many as 500 regulated stem cell lines derived from adult tissue, which could, Isacson says, be genetically matched to 75 to 90 percent of the population.
In their upcoming trial, Barker and his team of collaborators will implant fetal dopamine neurons into the brains of 20 patients in Europe and follow 130 other patients whose Parkinson's is progressing naturally. Learning from past procedural mistakes, the scientists have tightened the selection of patients, improved tissue preparation and placement, and rethought the length and follow-up for the multicenter trial. The TransEuro study is intended to provide proof of the principle that cell therapy can consistently repair the brain, Barker says. “The importance is the process, which we see as the stepping-stone to the next generation of cell-based therapies.”
Despite its theoretical superiority, populating the brain with new dopamine cells is not yet obviously better than existing treatments such as DBS, which brings faster results. In addition, other treatments in development may prove feasible. For example, in early 2014 researchers at Imperial College London reported promising results from the first gene therapy trials for Parkinson's patients. In this treatment, doctors insert genes for dopamine-producing enzymes into the striatum, a part of the midbrain that contributes to movement control.
Many researchers believe, however, that the remaining hurdles in producing and validating stem cell therapy can be cleared for Parkinson's. To Rao, who in his post at the NIH oversaw all the work under way in regenerative medicine, the progress so far has been encouraging. “These are the first steps in what could be a revolutionary treatment,” he says.