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STEM CELLS

Inside a Stem Cell Lab

A Young Scientist Committed to Scientific Progress

Human embryonic stem cell derived motor neuron SOURCE: Project A.L.S. While pundits and bloggers argue over the political implications of recent breakthroughs in stem cell science, Kathryn Hinsch visits one of the first privately funded stem cell labs and learns that research must continue on all fronts: embryonic, IPS, placental, and adult. Above, human embryonic stem cell derived motor neurons.

Certainty rings through most public statements on the stem cell debate, but its resonance is hollow, its very presence ironic. Certainty and science do not, as some policy leaders seem to think, waltz in perfect step. In fact, the opposite is true. Science begins with a question, proceeds through inquiry, matures through review and challenge, and progresses through innovation. Certainty, on the other hand, is static.

Three weeks ago, a new chorus of certainty found voice as teams of scientists in the United States and Japan announced they had genetically modified skin cells to demonstrate some characteristics of embryonic stem cells—using a method that involves no destruction of human embryos. But lost in the gleeful cacophony of the religious right’s pundits and bloggers as they subvert these discoveries into calls for yet more restrictions on free scientific inquiry are the voices of researchers who continue to work quietly and thoughtfully to uncover the causes of and cures for disease.

A five-minute discussion with any bench scientist involved in embryonic stem cell research will easily banish any certainty that human cloning is just around the corner or that potential babies are being destroyed. Every day in this country, stem cell scientists work on projects the fear mongers will never mention.

A Community Committed to Scientific Progress

One of those quiet voices belongs to Gist Croft, a young scientist working at Project A.L.S. in New York. In 1998 Valerie, Meredith, and Jenifer Estess founded Project A.L.S. when doctors diagnosed Jenifer with Amyotrophic Lateral Sclerosis, a fatal neuromuscular disorder, which causes progressive paralysis of the muscles (Jenifer died in 2003 at age 40). Faced with no central clearinghouse for information about the disorder, the sisters harnessed their energy, frustration, and friends, determined to bring the best science to ALS patients in pursuit of effective treatments and, ultimately, a cure. Their mission—to put a human face on cutting edge research—was a perfect match for Gist Croft’s commitment to values-driven science.

A Ph.D. candidate in neurobiology and behavior at Columbia University, Croft was not always sure he would end up in a lab. During his undergraduate years at Williams College, he bolstered his chemistry and molecular biology major with extensive course work in literature, political philosophy, and religious studies. “I come from a family of physicists, chemists, and doctors, so science was kind of a natural thing for me to rebel against,“ he admits. “Work in the humanities allowed me to come to science on my own terms.”

Before attending Columbia, Croft completed a postgraduate internship at Merck Pharmaceuticals, and then worked at Rockefeller University—where he studied how hormones and sickness affect the production of new cells in the brain. Those were exciting years for neurobiologists. Stem cells had just been defined and developed for research.

At Project A.L.S., the fluidity of scientific inquiry is not limited by an external agenda.

“The leap from mouse to human stem cells presented an incredible, exciting opportunity to combat our own disease, dysfunction, and injury,” Croft recalls, “almost a romantic pursuit: a way to combine the technical, philosophical, and intellectual challenges of science with something that has a true impact on human life and human health.” Finally, he’d found the marriage of science and literature he’d sought for years. “Honestly, I have to look back to Camus’ The Plague—a book I read as a young teen—for a description. The work became an existential commitment, a chance to do battle with public health challenges.”

Croft chose Columbia for its community of neurobiologists, who collectively studied the basics of synaptic transmission and cell differentiation and diversity. These researchers, at the apex of what has been dubbed the Decade of the Brain, were looking not only at learning and memory but also at neurological disease. Other scientists were working on mouse embryonic stem cell-derived models of development and disease, and some wanted to begin work with human models.

Gist Croft at the Project A.L.S. labGist Croft at the Jenifer Estess Laboratory.

Croft shared this interest. But as he discovered, the barriers were substantial. The 1996 Dickey amendment banning federal funds to any “research in which a human embryo or embryos are destroyed, discarded or knowingly subjected to risk of injury or death” had been dramatically reinforced by the Bush administration’s decision to withhold federal funds from scientists working on any stem cells lines derived after August 9, 2001. Fortunately, as Croft paraphrases, “Adversity can be the mother of invention. When I was ready to join a lab, my mentors Chris Henderson and Hynek Wichterle, Columbia professors and scientific directors at the Project A.L.S. lab were already working to set up this independent facility, able to conduct research outside the National Institutes of Health’s funding constraints.”

At Project A.L.S., the fluidity of scientific inquiry is not limited by an external agenda. Croft says he was drawn to the organization’s “vision to recruit scientists into productive collaborations that cross the boundaries of disciplines and institutions and move us beyond narrow views of professional self-interest.” On a given day, in addition to nurturing the cells through various stages of differentiation, Croft and his colleagues often confer with like-minded researchers at other institutions. This open exchange of ideas, this willingness to defer certainty and to crosscheck results, is essential to true scientific progress.

The Mechanics of Stem Cell Research

But what, exactly, do stem cell researchers do? Pervasive misinformation—or perhaps an absence of good information—about the mysteries of the stem cell research laboratory is strangling public debate. A brief glimpse of Croft’s work, even though it is only one of many specialized projects underway worldwide, reveals not some fearsome mad-scientist scheme, but rather painstaking scientific inquiry driven by reasonable hope.

It is telling that both scientists who created induced pluripotent cells plan to continue their work with embryonic stem cells in the face of their breakthroughs.

About four or five days after an egg is fertilized in vitro, this one cell develops into a blastocyst, a hollow microscopic ball of cells still unable to implant itself into the uterine wall. At one end of the hollow cavity lies the inner cell mass, a group of about 30 cells. In normal development, these 30 identical cells will ultimately give rise to every cell in the body. To derive an embryonic stem cell line, scientists remove this inner cell mass and transfer it to a culture medium. These cells have two defining characteristics, which define them and make them invaluable research tools: self-renewal and pluripotency.

A self-renewing cell will divide, over and over again, into an identical copy of itself. This means that once a stem cell line is established it provides an unlimited source of cells for research. A pluripotent cell can generate more than one type of offspring cell—in the case of embryonic stem cells, this means the ability to generate each of the ten thousand cell types in the body. But pluripotency is more complicated than self-renewal; it comes in degrees.

Stories have appeared in the media recently touting the wonders of adult stem cell research, and while adult stem cells are technically pluripotent, their germinating ability so far is type-limited. For example, an adult stem cell in the skin can produce several kinds of skin and hair cells, and an adult stem cell in the lateral ventricle of the brain can generate neurons for olfactory circuits, and some glial cells. But a skin cell cannot become a brain cell.

Articles in recent issues of Science and Cell have announced the creation of “induced pluripotent stem cells” (IPS) —which mimic embryonic stem cells—from ordinary skin cells, but the methods require viral delivery methods and genetic modification, techniques that raise additional ethical quandaries and increase the chance for error and mutation. The methods used can cause random disruptions to the new cells’ DNA, disruptions that can complicate controlled experimentation or even cause cancerous changes. An embryonic stem cell, on the other hand, carries within it the natural ability to generate any type of cell in the adult body without genetic modification or the use of retroviruses. It is telling that both scientists who created induced pluripotent cells plan to continue their work with embryonic stem cells in the face of their breakthroughs.

Building New Neurons

Embryonic stem cells, then, provide innumerable opportunities for research. To the scientists seeking a cure for ALS, this means turning embryonic stem cells into motor neurons. Motor neurons carry information between the brain and the muscles, messages that let us move at will and allow essential, involuntary movements like breathing, digestion, and swallowing. In ALS and related diseases like spinal muscular atrophy, the motor neurons begin to die. As the electrical impulses between brain and muscles cease, patients gradually become paralyzed muscle by muscle, losing both voluntary and involuntary function.

Past work on mouse embryonic stem cells determined that two factors were necessary and sufficient to direct an embryonic stem cell to differentiate into a motor neuron. The first is vitamin A, which tells the cells to take the next step on toward the spinal cord rather than settling in closer to the head. The second, a protein with the fanciful name Sonic Hedgehog, binds to the cells and, at a certain concentration, instructs them to become motor neurons.

In 2002, Columbia University’s Hynek Wichterle successfully generated motor neurons from mouse ES cells by applying these two signals at the appropriate time. The Jenifer Estess Laboratory, a joint undertaking of Columbia University faculty and Project A.L.S., is the first privately-funded lab to focus exclusively on the study of stem cells to treat ALS and related motor neuron diseases. Because it does not accept federal funding and has an open-door policy, scientists at Harvard University, Johns Hopkins University, the Salk Institute, Memorial Sloan- Kettering Cancer Center, and other New York-based institutions can collaborate with Columbia University-based scientists and clinicians. In this environment, Croft—and stem cell research—have flourished.

“Every day starts with discussions of ‘where we are.’ We always have undifferentiated cells in the expansion process, as many as a billion at a time at different points in their journeys,” Croft explains. “On day twenty-eight of differentiation, for instance, we need to confirm that the cells are following the path toward becoming motor neurons that we expect. In addition to doing what we know will work, we are always optimizing, trying to improve our results.”

The cells need care beyond application of the two chemical signals mentioned above. Like any living thing, they must have nutrients to survive: sugar, insulin, iron transfer proteins, fats, and vitamins. Their requirements change as they mature. For example, at one point in their development, motor neurons require a growth factor from their target, the muscles. The axons collect it and transport it back to the cells. In the absence of those target muscles, the scientists have to provide these proteins or the motor neurons will die. The cells also produce waste, and since they lack a way to remove the byproducts of their metabolism, scientists have to do it for them.

Why Do Cells Break, Anyway?

Mainstream understanding of stem cell research centers on substitution therapies, replacing damaged, diseased, or dead cells with healthy ones generated from embryonic stem cells: motor neurons for ALS, dopaminergic neurons for Parkinson’s disease, oligodendrocytes for multiple sclerosis, pancreatic islet cells for diabetes. But scientists are not content to fix things. They want to know why they break. This aspect of stem cell research receives little media attention.

The quest for cause is a primary goal of Project A.L.S. “Most instances of ALS are sporadic,” Croft says, “and we don’t know why. Do patients harbor a genetic mutation as yet undiscovered, or have their bodies been affected by environmental factors? While we’ve learned much from past models, they’ve always had one significant limitation: mice never get ALS. You have to do some genetic manipulation, insert into the mouse the human gene variant that leads to ALS. You’re always working once removed. Despite the enormous conservation across evolution, you can never eliminate the significant differences.”

Deriving new embryonic stem cell lines that are sick with one of the diseases these scientists are investigating would be an invaluable new research tool to study why diseases occur, how they progress, and on which to test drugs that might mitigate the course of disease. This approach is directly applicable to any inherited disease. The current political climate makes such research impossible for those who rely on federal funding. Dedicated scientists, however, are finding a workaround.

Human embryonic stem cell derived motorHuman embryonic stem cell derived motor neurons, shown at high magnification.

Croft and his colleagues at Project A.L.S. are taking small skin biopsy samples from ALS patients and expanding them in vitro. Once these samples are ready, researchers in Harvard molecular biologist Kevin Eggan’s lab will attempt to remove the genetic material from egg cells and replace it with the ALS material. In October, an article in Nature confirmed that this technique, known as somatic cell nuclear transfer (SCNT) produced stem cell lines from primate skin cells. “We knew SCNT worked in mice and other mammalian species, so we bet this day would soon come. Making it work with primate cells is a big step towards making it work with human cells.” The new results published recently on human induced pluripotent cells were more unexpected, and immediately posited an additional way to derive patient- and disease-specific stem cell lines. In either case, the goal is the same, as Croft explains, “If we can turn these disease- specific cell lines cells into motor neurons we will have a new tool to study how they become sick, the very mechanism of the disease. We will also, almost immediately, have a disease model on which to test or screen for potentially therapeutic drugs.” The only hurdles to using this approach for any given inherited disease are creating the disease specific cell line, and understanding how to generate in vitro the type of cells affected in that disease.

A Better Path to New Drugs

Given the vigor of public debate on health care costs, it is surprising that this third facet of embryonic stem cell research has not received more media play: it could drastically improve the drug development process, ultimately reducing the time drugs stay in the R & D pipeline and lowering consumer’s costs.

Imagine the drug development process as a funnel, with chemicals and cells at the large end and patients at the small opening. At the beginning, researchers investigate the effects of many different chemical compounds, usually on isolated enzymes because that process is easy to control. They could be working with a library of 100,000 compounds of myriad different molecular shapes and activities

Once they determine a positive, measurable effect, the drug leads are then tested on more sophisticated, animal cell-based models that represent some aspect of the target disease. With every round of elimination, the funnel’s opening grows smaller until those thousands of compounds have narrowed to, say, ten potential drugs. Those drugs are then tested on animals that, in most cases, have been inoculated with the target disease. After the initial testing and refinement process—which can take years—researchers are finally ready to administer the drugs to humans in clinical trials.

The difference between exact and close is a chasm in the controlled world of the laboratory.

But there is one catch. These drugs have never been proven on human cells. And drugs that work on animal models of disease can fail in human trials. If researchers could jump ahead and use embryonic stem cells at the level of cell-based testing, Croft notes, “they would know, at a very early stage, that drug candidates worked on the specific types of human cells affected clinically. Research costs and time could, accordingly, drop.” In addition, cell-based drug testing requires millions of cells. Using embryonic stem cells, a lab could generate millions of exact copies, without relying on closely replicated mouse cells. The difference between exact and close is a chasm in the controlled world of the laboratory. Thus, cell type specificity, species specificity, and unlimited numbers are important reasons for pursuing hESC research.

Cures Delayed Are Lives Lost

While each new scientific breakthrough has the potential to bridge that chasm, political posturing can only widen it. And as it grows, every citizen suffers. If policy suddenly restricts research to induced pluripotent stem cell projects, the gains of the last decade would at best require review, and at worst might be lost. Without question, cures would be delayed. And cures delayed are lives lost. Senator Arlen Spector (R-PA) told The New York Times, “My own view is that science ought to be unfettered and that every possible alternative ought to be explored. You’ve got a life-and-death situation here, and if we can find something which is certifiably equivalent to embryonic stem cells, fine. But we are not there yet.”

If the federal government continues to withhold funding from new embryonic stem cell projects, researchers could be forced to follow the money into nascent technologies that, while promising, offer no guarantees. Only when an enlightened administration lifts current restrictions can scientists collaborate, review each other’s work without jeopardizing their own funding, and continue to expand human knowledge.

It’s time to abandon the selective morality of partisan agendas and focus on the needs of the living. That’s what scientists like Gist Croft do, day in and day out, even as their hands are tied by the dictates of special interests. To win the battle against disease, dysfunction, and injury, stem cell research must continue on all fronts: embryonic, IPS, placental, and adult. Opponents of embryonic stem cell research, who base their objections on the sanctity of life, look the other way as, year by year, thousands of frozen embryos are simply discarded. Those embryos—otherwise bound for a biohazard container—could instead find life in the pancreas of a forty-year-old teacher with diabetes, in the central nervous system of a man slowly suffocating from ALS, or in the steady arms of a grandmother whose stable Parkinson’s disease now allows her to hold her newborn grandson. Sanctity lies in their lives, as well.

Kathryn Hinsch is the founder of the Women’s Bioethics Project and a member of the Science Progress advisory board.

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