A series of genetic markers sprinkled throughout the cells' chromosomes show the same pattern found in parthenogenetic mice as opposed to cloned mice, according to a report published online today in the journal Cell Stem Cell.
The result suggests that, although Hwang deceived the world about achieving the first human cloning, his group was first to succeed in performing human parthenogenesis, which may offer a way of creating cells that are genetically matched to a woman for transplantation back into her body to treat degenerative diseases.
"I think this is an extremely important—and solid—paper," says stem cell researcher Robert Lanza, vice president of research and scientific development at Applied Cell Technology, a regenerative medicine company headquartered in Alameda, Calif., who did not take part in the study. "It conclusively proves that the stem cell line in question was not cloned as claimed, but rather was generated through parthenogenesis."
The result follows on the heels of an announcement last month by another California stem cell company, International Stem Cell Corporation (ISC) in Oceanside, that it had successfully achieved human parthenogenesis for the first time. Last year, Italian researchers claimed to have achieved the same feat but have yet to publish their results.
"The fact that this has now been achieved by two independent groups gives me a far greater degree of confidence," Lanza says.
The new finding brings a measure of closure to a story that first rocked the science world in February 2004, when Hwang and colleagues at Seoul National University announced they had cloned a female donor's cell by transferring its nucleus into one of her egg cells stripped of its nucleus in a procedure known as somatic cell nuclear transfer (SCNT), and harvested embryonic stem cells from the resulting fusion. They published the result the next month in Science.
The claim went up in smoke in January of 2006 after a probe by the university concluded that Hwang had fabricated the evidence, which followed a similarly damning assessment of a landmark paper from the previous year in which the group falsely reported creating 11 cell lines genetically matched to their donors.
A cloned cell should be identical to its donor, but the probe found that of 48 common genetic variations, or markers, present in the 2004 cells, eight did not match their apparent donor. Investigators raised parthenogenesis as the most likely explanation but could not be certain.
Later, during a chance discussion with European colleagues, stem cell researcher George Daley of Children's Hospital Boston and the Harvard Stem Cell Institute learned that they had received samples of the cell line before the work was retracted. "We had read the suspicions that the cell was a parthenote, but also realized that it had never really been proven," Daley says.
To settle the case, they analyzed the genetic sequence of the cell line at 500,000 locations across the genome.
The DNA of any two people will differ on average at one of every 1,000 subunits, or base pairs, Daley says. When a chromosome from a sperm cell joins with that of an egg, these single nucleotide polymorphisms (SNPs or "snips") tend not to match each other.
The same goes for cloned cells. But in contrast, pairs of matching chromosomes in parthenogenetic cells tend to match one another in the middle and differ near the ends because of a genetic mixing process called recombination. In their paper, Daley and colleagues report that the SNPs in the Korean cell line do indeed match toward the center of the chromosomes, similar to five parthenogenetic mouse cell lines that the team created for comparison.
In a separate analysis, they also found that three regions lacked the chemical modifications, or imprinting, that paternal genes impose on a fertilized embryo to prevent those genes from being activated.
Jeffrey Janus, president and director of research for ISC, agrees that "Dr. Hwang's cells have characteristics found in parthenogenetic cells" but remains cautious, saying "it needs more study."
The Irony of It All
Stem cell experts say that Hwang and his team probably had no clue what they had achieved, because if they had they would have claimed credit for it.
"I think this is every bit as exciting as the SCNT they were claiming," says stem cell researcher Kent Vrana of Pennsylvania State University, who pioneered parthenogenesis in monkeys. "Parthenotes by their very nature are nonviable embryos, so you're not destroying embryos, which has some ethical advantages."
Vrana says the Korean team used a procedure common in attempts to induce parthenogenesis and SCNT alike, in which they injected egg cells with calcium and a protein synthesis inhibitor to mimic what happens when sperm fertilizes an egg.
To achieve SCNT, they first had to extract each egg's DNA and then inject the donor cell nucleus. Daley says the Korean scientists must have inadvertently left the DNA intact in one of the 242 egg cells they injected. "They claimed they verified the removal of the DNA,'' Daley says, "but obviously they didn't."
The injection of the donor nucleus could have failed if the injecting needle pulled it back out when withdrawn from the egg or if the egg somehow rejected the introduced nucleus, Vrana says.
Hwang's group purported to rule out parthenogenesis as an explanation in part by showing that two genes normally activated by paternal DNA were inactive in the cells. But Daley says such experiments are easy to misinterpret and are less conclusive than sequencing SNPs.
"I think they were just so blinded by what they hoped to accomplish, they missed it," Vrana says.
As a result, in late June, more than a year after Science retracted the 2004 paper, researchers at ISC were able to claim the discovery of human parthenogenetic cell lines as their own in the journal Cloning and Stem Cells. The group reported growing multiple parthenogenetic embryonic stem cell lines by incubating eggs in a warm, low-oxygen culture medium.
Before today's announcement, the work was already "awfully convincing," Vrana says, and surprisingly successful: out of some 50 donated eggs, the company grew six cell lines. Parthenogenesis in monkeys typically works only once every 90 eggs, he says.
Banking on Parthenotes
The therapeutic potential of parthenogenetic cells remains to be seen. The lack of imprinting from the paternal DNA may cause the cells to behave abnormally as they develop. Furthermore, they must have matching immune proteins to be transplanted back into a donor.
In principle, tissue banks of parthenogenetic cell lines could include enough different immune protein combinations to treat up to half of the U.S. population—men as well as women—Lanza says. But he adds that if human parthenotes routinely contain as many genetic mismatches as the Korean cells, the number of eggs needed to create such a bank could be prohibitively large.
Daley says his group hopes to acquire donated eggs from women with inherited diseases and use parthenogenesis to create cell lines to study those disorders. In the future, researchers will have to determine whether similar cells are safe and effective when transplanted.
"We're a long, long way," Daley says, "from realizing therapeutic uses of these cells."