28 APRIL 2006 VOL 312 SCIENCE www.sciencemag.org516

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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 experi-

ments—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.

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 Insti-

tute 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 Med-

ical 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 gov-

ernment 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 Med-

ical 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

Picking Up the PiecesAfter Hwang

Several groups around the world are trying to do what

Woo Suk Hwang fraudulently claimed to have done

After the fall. Woo Suk Hwang and his colleagues do not have the stem cells they

claimed to have made from cloned human blastocysts. CRE

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molecular processes that drive these diseases.The group will rely on so-called compassionatedonors, women who are willing to donate oocytesspecifically for research. Eggan and his col-leagues hope that using fresher, healthier oocytesthan those left over after in vitro fertilization(IVF) procedures will increase the chances ofsuccess. Hwang and his colleagues reported thatfreshly harvested oocytes from women youngerthan 30 were significantly more efficient thanoocytes from women 30 or older. That claim isplausible in light of well-established fertility sta-tistics, say researchers, but can’t be completelytrusted. Harvard researchers have said they hopeto attract women younger than 30 as donors.

Two other U.S. groups, in New York City andSan Francisco, say that for their first efforts theywill rely on excess oocytes from women under-going fertility treatments. One of the team lead-ers, fertility expert and developmental biologistRenee Reijo-Pera of the University of Califor-nia, San Francisco (UCSF), had planned to sendstudents to Seoul to learn Hwang’s techniques.

With those plans scotched, the team has a proto-col under review at the university that would useoocytes collected for IVF treatments but whichfailed to fertilize in the culture dish. Suchoocytes are likely to be lower quality, but theywould otherwise be discarded, so the ethicalquestions surrounding their use are less trou-bling. “We are still at a stage where the technol-ogy [for human SCNT] has not been properlydeveloped,” says Arnold Kriegstein, director ofUCSF’s stem cell biology program. Untilresearchers know more about which techniquesmight work best, he says, they will avoid treatingvolunteers with the ovary-stimulating drugsrequired for egg donation, which can cause seri-ous complications. The work is being funded byprivate donations.

The lab of developmental biologist LorenzStuder at Memorial Sloan-Kettering Cancer Cen-ter in New York City was one of a handful thatwas working with several cell lines from Hwang’slab when the scandal broke. Investigators laterdetermined that the lines were most likely notcreated through cloning but arose either fromearly parthenogenetic development, in which anunfertilized oocyte begins dividing, or from

IVF-derived embryos. Studer, who says he has notheard from Hwang since fraud allegations werefirst raised, will now collaborate with colleagues atRockefeller University and Weill Cornell MedicalCenter. The three institutions received a $50 mil-lion grant from the Starr Foundation in New YorkCity last year to focus on stem cell research, part ofwhich will fund nuclear transfer to create cell linesfrom ALS and Parkinson’s patients.

Studer cautions, however, that successfulcloning attempts may be few and far between. “Idon’t doubt that you can do it, but the efficiencymight be so low that you couldn’t do it on a practi-cal level,” says Studer, who hopes to use ES celllines for both basic research and drug screening.“It looks like the most likely efficiency is 10 timeslower than [Hwang and his team] claimed” lastyear—which might mean a success rate of one outof more than 200 tries.

In Europe, at least three groups have saidpublicly that they hope to get human cloningworking in their labs. All are being funded atleast in part by government grants. A group ledby Ian Wilmut of the University of Edinburghand Christopher Shaw of King’s College Londonreceived a license from Britain’s HumanFertilisation and Embryo Authority in February

2005 to conduct human nuclear transfer experi-ments, but Wilmut says the scandal hasprompted them to rethink their plans: “It wasnecessary to spend some time unlearning somethings that we thought we had learned fromHwang’s research.” The researchers are nowpreparing a new application for permission andfunding for a slightly different approach to cre-ating ES cell lines from Parkinson’s and ALSpatients, he says. The researchers may attemptto use rabbit instead of human oocytes, he says.(Researchers in China have reported derivinghuman ES cell lines from embryos generatedthrough SCNT using rabbit oocytes.)

After the Hwang debacle, researchers at theUniversity of Newcastle upon Tyne in theUnited Kingdom hold the distinction of havingpublished the only paper on human cloning thathas not been discredited. Alison Murdoch,Miodrag Stojkovic, and their colleaguesreported in 2005 in Reproductive BiomedicineOnline that they were able to create a singlehuman blastocyst, although they could notderive ES cells from it. Murdoch declines todiscuss recent progress until the team is readyto publish another paper.

Stojkovic has since moved to Valencia,Spain, where he is deputy director at thePrince Felipe Research Centre, a $180 millionfacility funded by local and national govern-ments and private sources. In March, theSpanish government legalized human nucleartransfer experiments; Stojkovic is now seek-ing approval from a national ethics committee.He says his team could start working withhuman material as early as this summer.

Stojkovic says he will obtain oocytes from alarge fertility hospital in Valencia that manages3000 cycles of fertility treatment per year. But hesays he won’t bother with leftover oocytes thatfailed to fertilize in the lab: “From what I haveseen, the potential [of fail-to-fertilize oocytes] isequal to zero. We need fresh human eggs. Whatyou get left over from the IVF clinic is notviable.” In fact, he says, every minute counts. Inthe paper describing the cloned blastocyst, heand his colleagues reported that oocytes weremost effective if they were enucleated within anhour after collection. He says he hopes to findwomen who produce significantly more oocytesthan they need or who would be willing to donatesome of their oocytes in exchange for a discounton the cost of their fertility treatment.

Finally, a team at the Chinese Academy ofSciences’ Shanghai Institutes for BiologicalSciences is now seeking approval for humancloning. “Hwang’s work was fake, but someonehas to do the real thing,” says Guotong Xu,deputy director of the Institute of Health Sci-ences there. The stumbling block is not likely tobe approval, says Xu, but money, as no oneknows whether China’s funding agenciesconsider human SCNT efforts worthwhile.

As the field attempts to rebuild post-Hwang,Studer hopes the groups will behave like infor-mal collaborators rather than rivals. “It is impor-tant that we all stay in contact … so we knowwhat we are each trying to do,” he says. Oocytesare scarce enough that teams should try to wasteas few as possible—and should avoid directlyduplicating each other’s work, he says.

Stojkovic says he is optimistic that someonewill soon succeed where Hwang and his col-leagues failed. “I have no doubt that soon some-one will have cloned human stem cells,” he says.“I don’t know any technical, biological, or ethi-cal reasons we should not continue.”

–GRETCHEN VOGEL

With reporting by Dennis Normile.

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Best yet. Miodrag Stojkovic and Alison Murdoch

and their colleagues generated this cloned human

blastocyst but were not able to derive ES cells from it.

Somatic Cell Nuclear TransferSomatic Cell Nuclear Transfer

Somatic cell Oocyte Remove

oocyte nucleus

Fuse somatic cell with

enucleated oocyte

Four-cell-stage

embryo

Prompt oocyte

to begin dividing

One week later …

Inner cell mass

Blastocyst-stage embryo

Culture inner cell mass Embryonic stem cell line

Derivation of Embryonic Stem Cell Lines

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Picking Up the Pieces After HwangGretchen Vogel

DOI: 10.1126/science.312.5773.516 (5773), 516-517.312Science

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