Therapeutic Cloning Research Paper

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Scientists at Harvard and the affiliated Children’s Hospital Boston announced on June 6 that they had begun experiments with somatic cell nuclear transfer (SCNT). By combining donated human eggs with skin cells from patients who have sickle-cell anemia or diabetes, the scientists will attempt to clone diseased cells and then to derive stem-cell lines from those cloned cells. If successful, the efforts will result in stem-cell lines that carry specific diseases, and thus rapidly accelerate research into causes and cures.

Because of the highly charged ethical and political debate over the work, scientists George Q. Daley ’82, M.D., Kevin Eggan, and Douglas Melton received permission to pursue the research only “after more than two yearsof intensive review by eight different institutional review boards and stem-cell oversight committees at five different institutions,” said University provost Steven E. Hyman. “This highly unusual collaborative effort of the Harvard Stem Cell Institute,” he noted, “involves researcher sat Columbia University Medical Center’s Naomi Berrie Diabetes Center, Boston IVF [a center for assisted reproduction], the Center for Reproductive Medicine at Brigham and Women’s Hospital, and the New York Stem Cell Foundation.” Because of the Bush administration ban on federal funding of stem-cell lines created after August 9, 2001, “there is no taxpayer money being used for any embryonic stem-cell work,” Hyman added. “This is all [made possible by] private donation.”

For his research into diseases of the blood, Daley, of Children’s Hospital Boston (and an associate professor of medicine at Harvard Medical School), will use frozen, failed-to-fertilize eggs donated by couples who have ended their fertility treatments. Faculty of Arts and Sciences professors Eggan and Melton, tackling diabetes, will instead seek donations of fresh eggs. “We don’t know which strategy will work in the long run,” Daley said. For example, the researchers anticipate, but don’t know, that fresh eggs may be harder to come by; previously frozen eggs, on the other hand—those that failed to fertilize during assisted reproductive efforts—might not function. By taking “separate but complementary approaches and by working together and communicating,” Daley said, “our hope is that we will actually answer these questions.”

Having performed somatic cell nuclear transfer successfully with mous embryonic stem cells (above), Harvard researches are now starting to work with human cells.
To ensure that egg donors' consent is fully informed, the Harvard Stem Cell Institute created a handbook for donors. Freshly donated eggs, rather than frozen ones, may be necessary for successful nuclear transfer.

Although no one has yet published a credible paper reporting the derivation of a human embryonic stem-cell line from a blastocyst created by nuclear transfer, the technological hurdles appear surmountable. A group in the United Kingdom has reported success with SCNT using fresh eggs, but made no attempt to derive a stem-cell line from the resulting cell mass, which was destroyed instead as part of a definitive proof that the nuclear transfer had succeeded. Melton, in an earlier collaboration with Boston IVF and the laboratory of Andrew McMahon, Baird professor of science, has created more than 30 human em- bryonic stem-cell lines for free distribution to scientists around the world. Separately at least, then, the two halves of the problem Harvard scientists now propose to tackle have been solved (see box). Successfully bringing them together would create a powerful new tool against diseases. Because stem cells are self-renewing once isolated in a petri dish, scientists would be able to set out thousands of samples of diseased cells and then test different drug compounds on each sample to see if one cured the disease.

Stem-cell scientists (from top) Douglas Melton, George Q. Daley, and Kevin Eggan.

Another advantage of such stem-cell lines is the opportunity they present for “homologous recombination”: gene therapy in a petri dish. Daley has already used this technique to successfully restore partial immune function in a mouse with severe blood disease. In that experiment, he cloned a cell from the mouse’s tail, derived a stem-cell line, repaired the defective DNA in a petri dish, verified that it had been fixed, and then put corrected cells back into the mouse, where they reconstituted its blood and immune system. As a practicing physician, he hopes one day to use this therapeutic cloning technique to cure his patients.

The diseases the scientists hope to study, such as diabetes and Parkinson’s, are frequently the result of complex interactions among multiple genes and the environment. That means “it is impossible in the laboratory to use established molecular techniquesto make cell lines or animal models for [study],” said Eggan. “In fact, it is difficult even to identify [such] patients using genetic methods until they step into the clinic with the disease. This is one of the only methods we can imagine using for making cell-based models of these diseases.”

Opponents object to such work on ethical grounds: they believe it involves the destruction of an embryo that could become a person. That is scientifically debatable, because a sperm-fertilized egg starts dividing immediately and thus has the capacity for self-directed growth, whereas cloned cells are coaxed into cell division by scientists using chemical or electrical stimuli.

Of much greater ethical concern to the oversight committees that reviewed the Harvard team’s proposal was egg donation (which clouded the now discredited therapeutic cloning work of South Korean scientist Hwang Woo Suk). Egg donation involves a series of carefully timed injections with three different hormones, sexual abstinence for a month, frequent visits to a nurse, and a surgical procedure under anesthesia to retrieve the eggs. To avoid even the appearance of improper inducement, egg donors will not be compensated, except for their expenses. And to ensure that their consent is fully informed, the Harvard Stem Cell Institute has created the Research Egg Donor Information Book to explain the research and the medical risks. Who might be willing to donate? “At this point,” said Eggan, “we might guess that women [whose family members] are afflicted with diseases that we are interested in studying might step forward to participate. That is our best hope.”

Therapeutic cloning is a two-step process. Scientists must first transfer the nucleus of a skin (or other somatic) cell that they want to clone into an enucleated egg (one that has had its nucleus—hence all its DNA—removed). This is extremely difficult to execute successfully: in effect, it is performing surgery on a single cell. The second part of the process, derivation of a stem-cell line from the new cell created by the nuclear transfer, is also challenging. Scientists coax the cloned cell to divide for several days until they have a mass of four to 50 undifferentiated cells. Stem cells must be harvested from this cluster, called a blastocyst, at just the right moment; researchers have about a 24-hour window during which this fleeting cell-type can be successfully transferred, to be grown indefinitely in culture on a bed of feeder cells.

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