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Work on unfertilized eggs gets company noticed

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Alone after losing her husband and daughter in a Russian flu epidemic, 60-year-old Elena Revazova came to California in 1997 and began looking for distant relatives.

Revazova had been the chief scientist at Russia's national cancer institute. But in the United States she lived in obscurity, taking a volunteer research job at the University of California Los Angeles veterans hospital because she figured no one would hire a 60-year-old woman with poor English, despite two Ph.D.s and a medical degree.

A decade later, Revazova's work is back in the scientific spotlight. An Oceanside, Calif., company she helped start, International Stem Cells, gained worldwide attention with the publication last summer of work done by Revazova and her team of scientists, who coaxed unfertilized human eggs to produce embryonic stem cells.

The company showed that the embryonic stem cells could be grown into more human embryonic stem cells, as well as differentiated into some of the 200 different cell types in the body.

That work, scientists said, could provide a source of human embryonic stem cells that sidestep the ethical debate swirling around the cells. It could also provide a source of stem cells that would not provoke a negative immune response when injected into humans - at least in women who provide the eggs.

"It's a big deal, it's a very nice advance," said Kent Vrana of Pennsylvania State University, when the article was published online in the journal Cloning and Stem Cells. Vrana had done similar work in monkeys.

The company is the first to intentionally create these so-called human parthenogenetic cells - though another article published last summer suggested that Korean stem cell researcher Woo Suk Hwang may have created parthenogenetic cells when he falsely claimed instead to have cloned human embryonic stem cells.

International Stem Cell is hoping to create a bank of these parthenogenetic stem cells that can be used by researchers around the globe, and to use cells to create new therapies for diabetes and diseases of the eye and liver.

This month, a scientific journal article by the company showed that they turned the embryonic stem cells into cornea tissue.

The company, which went public in January through a reverse merger with an inactive company, has been selling shares over the counter.


The founding of the company goes back to Revazova volunteering at the VA hospital.

Dr. Gregory Keller, a plastic surgeon and scientist in Los Angeles, had spread word that he was looking for a good scientist to work in his lab. He was contacted by the head of the lab at the UCLA hospital.

"He said 'we have a volunteer from Russia working in our lab, and I don't know much about her, but she's amazing,'" Keller recalled.

Apparently the lab had many difficult problems getting cells to grow and suddenly this Russian woman was able to make everything work, Keller said.

When Keller met with Revazova, she seemed surprised that he wanted to hire her. But like the other American scientists who worked with her, Keller was wowed by Revazova's work. In his small lab, they worked on growing fibroblast cells to repair vocal chords.

Over time, Keller learned more about her personal story.

Both her husband and daughter had been diabetics. The disease caused them to be immunologically impaired. When the Russian flu epidemic hit and medical supplies were in short supply for even an elite scientist's family, the two could not survive.

The loss fueled her interest in therapies for diabetes.

Keller brought Revazova together with William Adams, a financial expert he'd done some work with before who also had a personal interest in diabetes research. And they introduced her to Kenneth Aldrich, a venture capital specialist.

Together they decided to use Revazova's scientific skills as a basis for a company that would target therapies for diabetes.

And they recruited Jeffrey Janus, a scientist who was a member of the team that founded Clonetics Corp., a San Diego company that had been a leader in manufacturing human cells for clinical and research use.

Janus pulled together a scientific team that could work on two fronts for the company. One was research and development, including Revazova's work. The second was the creation of a cell-growing business that could earn revenue to support the research.

As Revazova researched diabetes, she became frustrated with the limitations of adult stem cells.

Adult stem cells can be derived from many different places in the human body. Unlike embryonic stem cells, adult stem cells do not require the destruction of a human embryo. But they are limited in what they can become, unlike embryonic stem cells, which evolve into the 200-plus different cell types in the body.

Revazova began looking at human embryonic stem cells but realized that even they, as a therapy, would have an inherent therapeutic problem - people who received a therapy made from stem cells with a foreign DNA would have immune rejection issues and be required to take immune suppressing drugs that have side effects.

So she began researching the possibility of coaxing an unfertilized human egg to create embryonic stem cells. These so-called parthenogenetic cells already exist in nature, Revazova explained recently. For instance, unfertilized bee eggs produce the male, worker bees. The fertilized eggs produce the female, queen bee, she said.

And scientists had simulated that process in animals.

She thought that by stimulating the human eggs chemically, and then controlling the temperature and oxygen in the environment in which they are incubated, they could be coaxed to live and mature for up to seven days and become a blastocyst, a cluster of about 200 cells. Within the blastocyst's inner cell mass are embryonic stem cells.

But any work Revazova wanted to do on human embryonic stem cells was made problematic because of funding restrictions that President Bush placed on the research, Janus said.

Revazova returned to Russia in March 2002, to work on the controversial cells with funding from the company. In her homeland, she could work unfettered by U.S. restrictions. And she knew many top-notch scientists who were hungry for work because they could not get sufficient funding from the government since the dissolution of the Soviet Union.

The researchers talked to hundreds of Russian women who had gone through in-vitro fertilization to have children, and as a result had leftover eggs, or oocytes, frozen in storage at IVF clinics. From the women who sought to donate their unwanted eggs to research, the researchers used 12 eggs, taking them only from people who had already successfully had children, Revazova said.

From them, they successfully created six new embryonic cell lines.

"What Elena did that was so important was that she did this repeatedly. It was not a one-time event," said Jeff Krstich, International Stem Cell's chief executive.

The efficiency with which the lines were created is also notable, scientists said. For an embryonic stem cell therapy to ultimately be successful, the cell lines will have to be created with efficient use of human eggs, which are not readily available.

Another possible advantage of the cell lines is that they may get around federal funding restrictions in the United States because they do not come from fertilized eggs, said Evan Snyder, who runs the embryonic stem cell research program at the Burnham Institute in San Diego.

"Of course, we'd have to make sure these cells can do everything they are supposed to do," Snyder said.

The company is now hoping to take advantage of its California headquarters, which gives it access to a growing pool of talented stem cell scientists and possible funding from the state's $3 billion taxpayer-supported stem cell research fund, Krstich said.


It appears its work in eye diseases has the potential to become its first product, since a third-party laboratory has certified that International's stem cells have become cornea tissue. Currently corneal tissue used for implant is taken from cadavers, and recipients have immunological rejection issues, Revazova said.

If the company's parthenogenetic cells can be used for a therapy, theoretically a woman's oocyte could be used to produce cornea tissue. Or, cornea tissue can be developed from a donor oocyte and statistically, at least, it would pose fewer rejection issues than tissue that comes from a fertilized egg and contains the DNA of two people, Janus said.

Hans Keirstead, a stem cell scientist at University of California Irvine, has been given some of the company's cells to evaluate and work with as an independent adviser.

While Krstich, the CEO, is optimistic the company's work in eye disease could be in human clinical trials within a year and a half with Keirstead's involvement, the UC Irvine doctor is more measured.

From his work on the cells so far, they appear to be parthenogenetic stem cells that differentiate, but Keirstead said he has no hard data yet.

Even without those results, the company's work is still a valuable contribution to the field because it is a new stem cell line, these are potentially autologous cells and there are not many people working on developing parthenogenetic cells, Keirstead said.

The advantage to having a business work on these lines is that it has financial backing to concentrate on them, so their chances of succeeding are higher than others, he said.

Some local scientists who have read articles about Revazova's work but have not seen the cells are excited by it. But they cautioned that more work must be done to investigate whether these cells can form tumors.

Snyder, from the Burnham Institute, also said there is still not enough known about embryonic stem cells to predict whether the absence of one set of parental genes is important.

"It could turn out to be important to have two sets of genes. With real fertilization you get two copies of a gene and one is silenced and one isn't. Sometimes you want the father's genes and sometimes you want the mother's version. It could be a problem to be stuck with one version," Snyder said.

Krstich said the real challenge for International now is securing another $1 million to bring the cornea project to market. Originally that project wasn't in the business plan, but developed as the scientists studied retinal disease.

A grant from the California Institute for Regenerative Medicine could be a possible funding source.

"The key for us is getting the money to get to trial," Krstich said. "Once we get to trial, getting money will be easier. Not easy, but easier."
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