From the contentious debate over federal funding for stem cell research, it would be easy to assume that if restrictions were lifted, research would blossom and miraculous therapies would spring up like mushrooms after a downpour. Those who have been following the controversy over federal subsidies know that even if funds were unrestricted, investigators would still have to clear several significant hurdles before treatments derived from human embryonic stem cells (hESCs) could become a reality.

DIFFERENTIATION

Pluripotentiality, the cells' greatest asset, also presents their most basic challenge: directing and controlling their differentiation into a specific cell type, generation after generation, in a consistent and predictable fashion. "Our ability to extract the cell type of interest is still in its infancy," says Douglas Kerr, MD, PhD, associate professor of neurology at Johns Hopkins University, Baltimore.

Currently, there are at least 2 ways of accomplishing this, explains Kerr. The first is to bathe the undifferentiated cells in a cocktail of growth factors, with recipes varying according to the desired cell type. Kerr and his colleagues used this technique to generate motor neurons from mouse embryonic stem cells, which they implanted into partially paralyzed rats. By suppressing the normal axon-inhibiting properties of myelin and administering glial-cell derived neurotrophic factor into the sciatic nerve, they were able to coax the stem cell-derived neurons to form synapses with the gastrocnemius muscle and achieve a 50% improvement in hind limb grip strength after 4 months.1

This approach does not work with all cells, including dopaminergic neurons, Kerr points out. The second method is to use transcription factors to overexpress a certain cell type, followed by sorting techniques to choose the cells when they start to express a certain phenotype.

Even then, successful differentiation in the limited quantities adequate for basic research is no guarantee that the same method will work in the long term on the commercial scale required for creating a viable human therapy. "You must be able to direct differentiation to one cell type day in and day out, and I don't think most groups have come to grips with that," says Steven Minger, PhD, director of the Stem Cell Biology Laboratory at the Wolfson Centre for Age-Related Diseases at King's College, London, and leader of the first group to develop an hESC line in the United Kingdom. The ability to proliferate over time with no loss of pluripotency or multipotency is one of the minimal requirements for a successful stem cell line, he explains.

TRANSPLANTED CELLS

The fate of the cells once they are transplanted presents another hurdle. "Would the cells survive and multiply and do what you want them to do after they are injected?" asks Thomas Swift, MD, professor emeritus and former chair of neurology at the Medical College of Georgia, Savannah, and president of the American Academy of Neurology (AAN).

The brain is a complicated organ: connections are continually being formed and cells are sprouting and dying. Some neurons in the brain have processes that are a foot long and extend into the spinal cord, Swift explains. Merely injecting stem cells into the brain is no guarantee that they will behave as desired. "You must reestablish the connections and re-create the anatomy. The single biggest question is: are these cells going to be able to reconstruct the anatomy necessary to restore function?"

"You cannot put a new cell into a brain that's been making synaptic connections for 30, 40, or 50 years," adds David Hess, MD, professor and current chair of neurology at the Medical College of Georgia. His own research involves implantation of adult stem cells that secrete factors that promote the growth and development of endogenous neurons and supporting blood vessels.

The potential for adverse effects is another issue that concerns stem cell researchers. An undifferentiated cell injected by mistake could give rise to a teratoma. Immune rejection is also a possibility. There is evidence that hESCs have an "immune-privileged" status, so they fail to elicit much of an immune response, at least in animals.2,3 But research in this area is still in an early phase, and more studies are needed to confirm these findings.

COMMERCIAL PRODUCTION: COMING SOON?

Even after the basic science is figured out, the challenge of churning out cells in the quantities required to make them commercially viable is formidable. At a stem cell conference in London last summer, Minger used the example of pancreatic islet cells for treating diabetes. The average pancreas has a million islets, each of which contains about 5000 cells, so one pa-tient would need approximately 5 billion cells. Multiply that by the millions of diabetic persons worldwide, and the scope of the problem becomes apparent.

One of the issues stem cell researchers are grappling with is the problem of developing suitable culture media. "Right now, very few lines are suitable for human use, because they are derived from systems that have used animal cells and other products. These may contain viruses or other pathogens that may elicit an immune response," Minger explains.

"Any treatment must be commercializable for use in patients," Hess points out. "Human embryonic stem cells are hard to expand and scale up in number without using animal proteins."

At least one company, Geron Corporation of Menlo Park, California, claims that it has developed proprietary methods for the growth, maintenance, and scale-up production of undifferentiated hESCs in culture media that are free of feeder cells, which would reduce concerns over immune rejection. Speaking at the same London conference at which Minger appeared, Thomas Okarma, MD, Geron's chief executive officer, reported that his company has developed scalable manufacturing procedures to differentiate hESCs to therapeutically relevant cell types. "We are now testing 6 different therapeutic cell types in animal models," he said. "In 5 of these cell types, we have preliminary results suggesting efficacy as evidenced by durable engraftment or improvement of organ function in the treated animals."4

The first hESC-derived cells Geron plans to test in humans come from a line of oligodendroglial progenitor cells developed to repair spinal cord injuries. In animal studies, "the cells behaved as if they were making a spinal cord for the first time," Okarma said at the conference. He predicted that clinical trials would begin in the first quarter of 2007.

Kerr, who has participated in some of the Geron research, says that 2008 or 2009 may be a more realistic projection.

Minger, however, believes it will be at least 7 to 15 years before hESC products are ready for commercial use in humans. "We have to learn how to transplant the cells and how much dopamine or insulin or other factors we are going to need. Our major problem is, can we make the cells?" He terms predictions that treatments could appear within 2 to 3 years as "grossly irresponsible," and adds, "when I'm ready to transplant these cells into my mother, that's when we'll be ready to use them clinically."

Sharing this view is the California Institute for Regenerative Medicine (CIRM), the body created in 2004 after the passage of Proposition 71, which approved $3 billion in state funds over 10 years for research on hESCs as a way of getting around federal funding restrictions. On October 4, CIRM stated in its newly released scientific strategic plan that it intends to help bring several promising therapies to clinical trials within the next decade, but that stem cell therapies for routine use were unlikely to appear before then.5

PATENT PROBLEMS

Yet another challenge to the widespread use of hESCs in the United States comes not from a scientific source but a legal one. In fact, some observers believe it has dampened hESC research in the United States as much as federal policy.

In 1994, Ariff Bongso, PhD, an embryologist at the University of Singapore, became the first person to successfully isolate hESCs from 5-day-old human blastocysts. He followed that up in 2002 by developing a method for culturing the cells in media free of animal products, thus clearing one of the most significant hurdles to clinical research.

While Bongso showed that hESCs could be isolated, he was unable to maintain a viable cell line. That was achieved in 1998 by James Thomson, PhD, a veterinarian and developmental biologist at the University of Wisconsin, Madison. The United States Patent and Trademark Office awarded Thomson 3 patents based on his research, but as a University of Wisconsin employee, he turned them over to the Wisconsin Alumni Research Foundation (WARF), a nonprofit group established in the 1920s to handle the school's intellectual property revenues.

Essentially, the patents give WARF the rights to all hESCs developed in the United States. Through them, the foundation claims the right to demand fees, royalties, and annual payments from commercial users of hESCs--not only from the lines developed at the University of Wisconsin but from virtually every hESC line available. WARF imposes fees ranging from $75,000 to $400,000, depending on a company's size and the terms of its license. Currently, Geron holds the exclusive commercial rights from WARF to nerve, heart, and pancreatic cells derived from hESCs.

"We would argue that WARF's position has driven scientists out of the United States as much as the federal funding issues," says John Simpson, stem cell project director for the Foundation for Taxpayer and Consumer Rights, a California consumer rights group. "It has a chilling effect on the exchange of scientific ideas, and it interferes with smaller companies being able to get venture capital funding for their research," he tells Applied Neurology.

The WARF patents could also prevent the public from benefiting from research that it has subsidized. For example, CIRM executives have stated they would like to receive royalties on research funded by the institute as a way of giving taxpayers a return on their dollars, but much of those funds would be wiped out if WARF insists on receiving its share. The Juvenile Diabetes Foundation has begun awarding grants to stem cell researchers in other countries, claiming it finds WARF's demands too onerous.6

Last July, Simpson's organization, along with the Public Patent Foundation, requested a formal review of WARF's stem cell patents on the grounds that the patents inhibit scientific enquiry. They argue that Thomson just took technology that had been described for isolating animal stem cells as early as 1983 and applied it to human cells. "Patenting embryonic stem cells is like patenting food just because you can cook," Simpson wrote in a commentary.6 In October, the patent office agreed to conduct a review of the patents' validity. A final decision could take several years.

ALTERNATIVES

Given the political, logistic, and financial impediments to hESC research, many investigators are trying to obtain the information they seek in other ways. Adult stem cells are seen as an alternative. Hess, for one, believes that many investigators underestimate their potential. In his research, models of animal stroke experienced at least a 25% functional improvement following the transplantation of 200,000 to 400,000 multipotent adult progenitor cells derived from bone marrow. Fewer than 1% of the transplanted cells were present in the animals 2 months later, but there was evidence that the rats had developed new neurons, apparently from endogenous stem cells. "There are ways around using embryonic stem cells, and if we took that route we'd be much further ahead than we are now," Hess maintains.

Other experts aren't so sure. "Adult stem cells are a powerful source, but they are not the blank slate mother-of-all-cells that embryonic stem cells are," says Kerr. "You can reprogram to a different fate, but it's very hard. We've tried."

A position statement on the use of stem cells in biomedical research published jointly by the AAN and the American Neurological Association states that "the potential for translating adult stem cell research into therapy is far more uncertain" than that of embryonic stem cells.7 "We don't know the potential of other types of stem cells, like hematopoietic or other adult cells," says Swift, one of the statement's coauthors. "They may have a very wide potential to do all kinds of things, even if it's not as much as embryonic stem cells."

Hess, however, believes that some investigators' political or ethical beliefs may be giving them tunnel vision. "There are alternatives to all of this," he says. "The question is, do we want to look for them?"

REFERENCES1. Deshpande DM, Kim YS, Martinez T, et al. Recovery from paralysis in adult rats using embryonic stem cells. Ann Neurol. 2006;60:32-44. 2. Li L, Baroja ML, Majumdar A, et al. Human embryonic stem cells possess immune-privileged properties. Stem Cells. 2004;22:448-456. 3. Drukker M, Katchman H, Katz G, et al. Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells. 2005;24:221-229. 4. Okarma T. Taking hESC-based products into the clinic. Lecture presented at: European Stem Cells & Regenerative Medicine Congress; June 7, 2006; London. 5. Foundation for Taxpayer and Consumer Rights. Stem cell institute plan shows refreshing candor consumer advocates say. Press release. October 4, 2006. 6. Simpson JM. The missing link in stem-cell research. Sacramento Bee. July 2, 2006. 7. American Academy of Neurology and the American Neurological Association. Position statement regarding the use of embryonic and adult human stem cells in biomedical research. Neurology. 2005;64:1679-1680.

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