Science 28 April 2006: Vol. 312. no. 5773, pp. 516 - 517

Science 28 April 2006: Vol. 312. no. 5773, pp. 516 - 517

News Focus, Gretchen Vogel

Several groups around the world are trying to do what Woo Suk Hwang fraudulently claimed to have done

A year ago, it seemed so easy. In May 2005, Woo Suk Hwang and his colleagues told the world that they could make embryonic stem (ES) cells from cloned human embryos with an efficiency that astounded--and thrilled--their colleagues. In roughly one out of every 12 tries, the South Korean team reported, they could produce ES cell lines that were a genetic match to patients. Scientists hoped to use such cells to probe the genetic triggers of diseases such as diabetes and amyotrophic lateral sclerosis (ALS). Some dreamed of using them as the raw material for developing new tissues and cells that could treat previously incurable maladies.

A few months ago, those claims famously unraveled. It is now clear that Hwang’s team does not have any ES cell lines created from patients. It is also clear that the group didn’t fail for lack of trying: The team apparently used more than 2200 donated human oocytes in their experiments--more than five times the number they claimed in their papers (Science, 10 February, p. 754). The meltdown dashed the hopes of researchers and patients around the world, leaving many wondering whether cloning might be too difficult after all.

But as the shock of the scandal wears off, a handful of groups around the world are trying to do what Hwang and his group apparently couldn’t. At least three groups in the United States, three in Europe, and one in China say they are preparing to start efforts to derive ES cells from cloned human embryos. In attempting this feat, they all face two substantial hurdles: a limited supply of human oocytes and a lack of data on how to use them most efficiently.

Miodrag Stojkovic and Alison Murdoch and their colleagues generated this cloned human blastocyst but were not able to derive ES cells from it

Most researchers agree that they have to discount nearly everything they thought they had learned from Hwang, but they also know that Hwang’s techniques did achieve some successes. The lab does have one confirmed--and unprecedented--claim: It cloned a dog. And investigators at Seoul National University concluded that the lab did produce cloned human blastocysts, or week-old embryos, in about one out of every 10 attempts. But the team apparently failed to derive viable ES cells from those cloned embryos. It is not clear whether the fault lies with low-quality embryos generated by cloning or with the techniques the team used to try to derive stem cells.

A collaboration at Harvard Stem Cell Institute is set to find out. Even before Hwang’s claims fell apart, researchers there were planning to try their hands at deriving human ES cells through a process known as somatic cell nuclear transfer (SCNT). A successful derivation involves two distinct steps, both of which require considerable skill. In SCNT, scientists remove the nuclear DNA from an oocyte, attempting to inflict as little damage on the cell as possible. They then fuse the enucleated oocyte with a skin cell or other somatic cell. The oocyte provides signals that reprogram the somatic cell DNA and enable it to direct the development of an early-stage embryo. To make ES cell lines, scientists next isolate the group of cells called the inner cell mass from week-old cloned embryos and coax them to grow in culture dishes.

Now, almost 2 years after they started, Douglas Melton and Kevin Eggan of Harvard University and George Daley of Harvard Medical School in Boston have accumulated nearly all the approvals and permissions they need to start accepting oocyte donations. The process has involved at least five ethics committees and Institutional Review Boards, which must review the ethical safeguards governing donations of oocytes and also of somatic cells from patients. Because current government rules prohibit the use of federal money to derive new human ES cell lines, the Harvard team is funding this effort--including the facilities--with money from the Stowers Medical Institute in Cambridge, Massachusetts, the Juvenile Diabetes Research Foundation International in New York City, and other private donors.

The Harvard team wants to create cell lines from patients with diabetes and ALS, which they hope will help researchers understand the genetic and molecular processes that drive these diseases. The group will rely on so-called compassionate donors, women who are willing to donate oocytes specifically for research. Eggan and his colleagues hope that using fresher, healthier oocytes than those left over after in vitro fertilization (IVF) procedures will increase the chances of success. Hwang and his colleagues reported that freshly harvested oocytes from women younger than 30 were significantly more efficient than oocytes from women 30 or older. That claim is plausible in light of well-established fertility statistics, say researchers, but can’t be completely trusted. Harvard researchers have said they hope to attract women younger than 30 as donors.

Two other U.S. groups, in New York City and San Francisco, say that for their first efforts they will rely on excess oocytes from women undergoing fertility treatments. One of the team leaders, fertility expert and developmental biologist Renee Reijo-Pera of the University of California, San Francisco (UCSF), had planned to send students to Seoul to learn Hwang’s techniques. With those plans scotched, the team has a protocol under review at the university that would use oocytes collected for IVF treatments but which failed to fertilize in the culture dish. Such oocytes are likely to be lower quality, but they would otherwise be discarded, so the ethical questions surrounding their use are less troubling. “We are still at a stage where the technology [for human SCNT] has not been properly developed,” says Arnold Kriegstein, director of UCSF’s stem cell biology program. Until researchers know more about which techniques might work best, he says, they will avoid treating volunteers with the ovary-stimulating drugs required for egg donation, which can cause serious complications. The work is being funded by private donations.

The lab of developmental biologist Lorenz Studer at Memorial Sloan-Kettering Cancer Center in New York City was one of a handful that was working with several cell lines from Hwang’s lab when the scandal broke. Investigators later determined that the lines were most likely not created through cloning but arose either from early parthenogenetic development, in which an unfertilized oocyte begins dividing, or from IVF-derived embryos. Studer, who says he has not heard from Hwang since fraud allegations were first raised, will now collaborate with colleagues at Rockefeller University and Weill Cornell Medical Center. The three institutions received a $50 million grant from the Starr Foundation in New York City last year to focus on stem cell research, part of which will fund nuclear transfer to create cell lines from ALS and Parkinson’s patients.

Studer cautions, however, that successful cloning attempts may be few and far between. “I don’t doubt that you can do it, but the efficiency might be so low that you couldn’t do it on a practical level,” says Studer, who hopes to use ES cell lines for both basic research and drug screening. “It looks like the most likely efficiency is 10 times lower than [Hwang and his team] claimed” last year--which might mean a success rate of one out of more than 200 tries.

In Europe, at least three groups have said publicly that they hope to get human cloning working in their labs. All are being funded at least in part by government grants. A group led by Ian Wilmut of the University of Edinburgh and Christopher Shaw of King’s College London received a license from Britain’s Human Fertilisation and Embryo Authority in February 2005 to conduct human nuclear transfer experiments, but Wilmut says the scandal has prompted them to rethink their plans: “It was necessary to spend some time unlearning some things that we thought we had learned from Hwang’s research.” The researchers are now preparing a new application for permission and funding for a slightly different approach to creating ES cell lines from Parkinson’s and ALS patients, he says. The researchers may attempt to use rabbit instead of human oocytes, he says. (Researchers in China have reported deriving human ES cell lines from embryos generated through SCNT using rabbit oocytes.)

After the Hwang debacle, researchers at the University of Newcastle upon Tyne in the United Kingdom hold the distinction of having published the only paper on human cloning that has not been discredited. Alison Murdoch, Miodrag Stojkovic, and their colleagues reported in 2005 in Reproductive Biomedicine Online that they were able to create a single human blastocyst, although they could not derive ES cells from it. Murdoch declines to discuss recent progress until the team is ready to publish another paper.

Stojkovic has since moved to Valencia, Spain, where he is deputy director at the Prince Felipe Research Centre, a $180 million facility funded by local and national governments and private sources. In March, the Spanish government legalized human nuclear transfer experiments; Stojkovic is now seeking approval from a national ethics committee. He says his team could start working with human material as early as this summer.

Stojkovic says he will obtain oocytes from a large fertility hospital in Valencia that manages 3000 cycles of fertility treatment per year. But he says he won’t bother with leftover oocytes that failed to fertilize in the lab: “From what I have seen, the potential [of fail-to-fertilize oocytes] is equal to zero. We need fresh human eggs. What you get left over from the IVF clinic is not viable.” In fact, he says, every minute counts. In the paper describing the cloned blastocyst, he and his colleagues reported that oocytes were most effective if they were enucleated within an hour after collection. He says he hopes to find women who produce significantly more oocytes than they need or who would be willing to donate some of their oocytes in exchange for a discount on the cost of their fertility treatment.

Finally, a team at the Chinese Academy of Sciences’ Shanghai Institutes for Biological Sciences is now seeking approval for human cloning. “Hwang’s work was fake, but someone has to do the real thing,” says Guotong Xu, deputy director of the Institute of Health Sciences there. The stumbling block is not likely to be approval, says Xu, but money, as no one knows whether China’s funding agencies consider human SCNT efforts worthwhile.

As the field attempts to rebuild post-Hwang, Studer hopes the groups will behave like informal collaborators rather than rivals. “It is important that we all stay in contact … so we know what we are each trying to do,” he says. Oocytes are scarce enough that teams should try to waste as few as possible--and should avoid directly duplicating each other’s work, he says.

Stojkovic says he is optimistic that someone will soon succeed where Hwang and his colleagues failed. “I have no doubt that soon someone will have cloned human stem cells,” he says. “I don’t know any technical, biological, or ethical reasons we should not continue.”