From the lab to the patient

Work with embryonic stem cells is in its infancy...

Why are new embryonic stem cells needed?

In 2001, President George W. Bush announced that the federal government would provide funds for human embryonic stem cell research, but

Going to the source

	Stem cell research slated to expand at Children's
	Photo: Mark Ostow

by Nancy Fliesler

If there's anything that represents pure potential, it's the stem cell," says Leonard Zon, MD, the Children's Hospital Boston researcher heading up the hospital's new Stem Cell/ Developmental Biology research program.

Like the acorn from which an oak will grow, stem cells are the starting point for a cell-division cascade that produces all the billions of special cells in skin, muscle, blood, brain, eyes, heart and other organs that make up the human body. The youngest stem cells, derived from embryos that are only days old, have the greatest creative potential. These so-called embryonic stem cells, the acorns, can make everything else, the oak. From the acorn, specialized stem cells arise to create the branches, and further specialized stem cells create the twigs, leaves and even more acorns. The same potential exists for the human body, raising the possibility of growing stem cells to replace all types of diseased human organs and tissues.

Embryonic stem cells are "pluripotent," meaning that they're able to grow any type of cell. Stem cells from older embryos or adults are "multipotent," meaning they can still grow various kinds of cells, but are "committed" to making cells of a specialized tissue such as the nervous system, the vascular network, or muscle. In other words, multipotent cells (also called "adult" stem cells) have somehow been told—or have decided—what their identity is going to be, and they proceed along that defined pathway to make a particular tissue or organ—say, the cells of the blood system. As gestation progresses, stem cells become more and more specialized.

Directly from the source
Because of their flexibility, embryonic stem cells are eagerly sought after by researchers. Experiments already show that they can be induced to grow into bone marrow, nerve cells, muscle and other tissues. These successes raise the possibility of an entirely new approach to treating disease, and research with embryonic stem cells is exploding worldwide. In the United States, however, federal rules prohibit using government money to create and experiment with new embryonic stem cells because of their source: embryos obtained from in vitro fertilization (IVF) procedures.

In IVF—made famous in 1978 with the birth of the first test-tube baby—the parents' eggs and sperm are brought together in the laboratory to optimize the chances of fertilization. If successful, the cells of the fertilized egg begin dividing, and the resulting mass of cells, now called an embryo, is implanted in the mother's uterus in the hope of achieving pregnancy. However, IVF often generates more viable embryos than are safe to reimplant (due to the risks of a multiple pregnancy). Parents then have the option to discard the unused embryos or freeze them for potential use in the future. Some choose a third option and donate the unused embryos to research. Many people object to such studies for religious or moral reasons, since they destroy embryos that have the biological potential to become human beings. As a result, government-funded research can use only existing human embryonic stem-cell lines—those created before President Bush's doctrine, announced in 2001. But scientists note that the government-approved lines have a number of limitations (see sidebar on page 20). Having a greater number and variety of lines will help ensure that scientists can successfully generate a particular tissue they need.

Children's is among a handful of institutions pursuing embryonic stem-cell research without federal funding, relying on support from private donors. In April, Children's launched its new interdisciplinary Stem Cell/Developmental Biology research program, and tapped Zon to guide it. Along with being an expert in blood diseases, he is one of Children's 10 Howard Hughes Medical Institute investigators, and is president of the International Society for Stem Cell Research, a nonprofit organization that promotes the dissemination of information and ideas relating to stem cells.

The Children's stem cell program, housed in the hospital's new Karp Family Research Laboratories, will consist of 35 to 50 dedicated researchers, plus over 30 associated faculty from other departments who are doing related research. These numbers are expected to grow over time. "We're trying to become one of the first groups to use stem cell therapy to cure childhood disease," Zon says. In addition to its own program, Children's will be an active participant in the new Harvard University Stem Cell Institute, whose executive committee Zon is chairing. "No place can compare with the scientific firepower at Children's and Harvard," he says.

At Children's, Zon is setting up a dedicated laboratory for human embryonic stem cell work that can be used by researchers throughout the hospital. The lab will provide resources for culturing embryonic stem cells and developing new lines to work with. The bigger goal, however, is to learn what genetic and molecular triggers drive embryonic stem cells to develop into various tissues, such as hormone-making cells, skin cells and muscle cells. Once these triggers are understood, scientists may be able to push stem cells to divide and form specialized tissues. Diabetes specialists, for example, could get new sets of insulin-making beta cells that could be added to the pancreas without being rejected. Neuroscientists could get a supply of healthy new neurons that wire themselves correctly into the brain, perhaps replacing those that die in Alzheimer's, Parkinson's and Lou Gehrig's disease, for example.

"We need to see what these cells can do," says Zon.

Lessons from blood
Children's researchers have long been probing the basic biology of stem cells, including adult stem cells. Research has shown that many mature tissues contain a residue of special adult stem cells that stand ready, like a rescue squad, to repair damage in case of injury. Bone marrow is the tissue best understood so far, but there also seem to be adult stem cells in the nervous system, skin, blood vessels and other organs. Research has even suggested that adult stem cells from one organ, such as bone marrow, can be coaxed into switching identity and growing cells for another system, such as muscles. But much of this research was flawed and has been discounted.

The current stem cell research program grew out of the hematology program under David Nathan, MD, Children's physician-in-chief from 1985 to 1995. As a result, Children's first expertise is in hematopoietic stem cells, which give rise to the various components of blood, such as red cells and immune-system cells. Zon, for example, is working with young zebrafish, whose transparent bodies let him watch as various blood components are put in place and observe what happens when genetic mutations are introduced.

On the clinical side, Children's has an active Stem Cell Transplantation Program, where blood stem cells taken from donor bone marrow are being used to treat such diseases as leukemia, other cancers of the blood system, severe aplastic anemia, and severe combined immune deficiency (SCID), which makes children vulnerable to every infection they meet. Unfortunately, using donated bone marrow is problematic since many children can't find a suitable donor that matches their tissue type. Without a proper match, they can develop graft-versus-host disease (GVHD), in which infection-fighting cells from the donor recognize the patient's body as being different or foreign and attack it. GVHD can be prevented by suppressing the immune system with medication, but this can open the door to infection.

George Daley, MD, PhD, associate director of the Stem Cell/Developmental Biology research program, is trying to avoid the need for donors altogether by tapping the potential of embryonic stem cells. Daley, who came to Children's in October 2003, is widely recognized as one of the nation's foremost stem cell researchers. He was one of the first four scientists to receive federal funding for human embryonic stem cell studies, and did groundbreaking work that paved the way for development of Gleevec, a drug that has been highly effective in treating chronic myeloid leukemia

He has a research group with about 20 people who are interested in blood development. "We're trying to address this problem of tissue donors by generating a line of universally transplantable cells," he says. "We're focused on embryonic stem cells and directing them into becoming adult blood stem cells."

A longer-term strategy is to customize stem cells so they exquisitely match each recipient's tissue type. "We could make a new stem cell line that is tailored to the patient," Daley speculates. "We can already do that in mice." To create such a line, the researchers would get a cell sample from a patient, take the nucleus from one of these cells, and then implant that nucleus into a stem cell from which the original nucleus had been removed.

It may be possible to replace a faulty gene—one causing sickle cell anemia, for example—with a normal, healthy copy of the same gene, Daley adds. Then, the repaired stem cells could be introduced into the body, allowed to divide, and used to reconstitute the sick child's bone marrow. It's a treatment that should last a lifetime.

"We are keenly interested in the genetic diseases of children," says Daley. "And the first zone of opportunity is going to be the intractable immune deficiency diseases." Some of these diseases are already treatable with gene therapy, but major problems arose in France recently when a few children given new immune-system genes—delivered by viruses—developed leukemia. That news seriously slowed gene therapy research, and has focused increasing interest on the idea of using stem cells as a way of correcting genetic defects.

The genetics of stem cells themselves is another area of intense study. The Children's program has a core laboratory dedicated to stem cell genomics, headed by Yi Zhou, PhD, and is one of several major centers participating in the large, NIH-funded Stem Cell Genome Anatomy Project. The NIH project's goal is to purify adult stem cells and find out what genes are expressed in stem cells from blood, liver, kidney, the gastrointestinal tract and bone. Zhou and colleagues are focusing on blood stem cells, comparing what genes turn on in zebrafish and mice to guide development of the different cells that make up blood.

Targeting diseases of young and old
While the Stem Cell/Developmental Biology research program has its roots in hematology, Zon plans to dramatically expand its scope. "We're one of the best groups in the world for blood stem cell research," Zon says. "We're now developing programs to take our blood stem-cell knowledge to other organs and other diseases."

Zon is currently conducting a search for two new faculty to join the program. The goal is for one of the new recruits to have expertise in either brain stem cells or pancreatic stem cells, while the other will focus on cancer: certain tumor cells have stem-cell-like characteristics, making them able to renew themselves endlessly; this makes them able to perpetuate the cancer and form new malignant tumors. Most other cancer cells lack this ability. Children's researchers will explore what genes are being activated in the self-renewing cancer cells, and make these the targets of therapy. "We'll be looking at cancer in a unique way, envisioning it as a stem-cell problem," says Zon.

He is also building a network of collaborators from all corners of the Children's research community. These researchers' interests range from developing stem-cell therapies to rebuild muscles ravaged by Duchenne muscular dystrophy to learning how the kidneys are formed in the developing embryo. Indeed, many developmental biologists, who study embryonic organ development, are offering to share resources with the stem cell program. "A lot of people are excited about the opportunities in having a stem cell center here," Zon says.

Researchers studying tissue engineering and regenerative medicine will also be tapped. Tissue engineers use stem cells to grow new tissues in the laboratory by "seeding" the cells onto scaffolds that give them shape and form, or getting them to implant at the site in the body needing repair. Vascular biologists, for example, are exploring the potential of certain adult stem cells of the blood system to rebuild damaged blood vessels and heart valves. Researchers in regenerative medicine are studying how mature cells can be made to defy nature and revert to a primitive state that resembles stem cells, and then re-specialize to regrow injured tissues—just as newts can grow new limbs. Elsewhere at Children's, neuroscientists are working to regenerate injured nerves, and researchers in otolaryngology are trying to treat deafness by regenerating hair cells in the inner ear.

These investigators' work has turned up many molecular and genetic clues that are likely to advance stem cell research. "The collaborations being forged at Children's Hospital Boston and throughout Harvard will be critical to the success of bringing stem cells into clinical medicine," says Zon. "Our basic biology is set up to push the field forward and ultimately translate the work to the clinic."

Children's is forging ahead into this new field of science, despite funding barriers. In the process, its researchers are working to meld stem cell biology into their ongoing studies of molecular biology, cell biology and human genetics. As they do so, they can be expected to play a defining role in stem cell research as medicine is, perhaps, changed forever.

The philanthropy factor
Never before has philanthropy been so critical to a scientific endeavor that has the potential to transform basic research into therapies for millions. Stem cell research holds the promise of helping patients suffering from illnesses as diverse as childhood leukemia and Parkinson's disease. Yet traditional funding sources are severely hindered, due to restrictions in federal funding for embryonic stem cell research and significant cutbacks for NIH grants. Moreover, prospects for industrial funding are limited by restrictive license terms proposed by patent holders for stem cell derivation. Visionary philanthropists can fill the gap and help bring about a pivotal medical breakthrough.