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SUMMER 2002
Reflections on Cloning
by Judy Bolyard Purdy

This isn’t the first time that advances in technology have led some to question whether scientists are playing God. But in the case of cloning, which inherently involves the very building blocks of life, perhaps such accusations were inevitable.

Steve Stice sees it differently.

“There’s a lot of concern that we’re producing monsters,” said Stice, a University of Georgia professor of animal and dairy science and one of the early pioneers in cloning research. “Nothing could be farther from the truth.

“I say we’re doing what Mother Nature allows us to do,” he said. “I try to get others to understand where I think cloning fits in.”

In Stice’s view, it fits squarely into the fields of agriculture and medicine, a tool to be used to produce more and higher-quality food and to manufacture cures in the war against disease.

Where it does not fit, he said, is in people. “I think cloning people is dangerous,” Stice said. “I don’t think it should be done. It’s morally repugnant to me.”

Since joining the UGA faculty almost four year ago, Stice has applied for six patents — four on cloning technology and two on developing stem cells into nerve cells. He already holds the first U.S. cloning patent, which is based on technology he helped develop a decade before the birth of Dolly, the sheep that became the first mammal cloned from an adult cell.

“I knew from the start the potential of cloning, what it could do some day. And I knew that stem cells had great potential,” he said. “Really the thing that drives me is seeing what these technologies can do in the real world. I’d like to take basic technology and move it into applications so people can use it.”

Farm, "pharm" and medicine

Perhaps the most rapidly developing applications are in agriculture. Stice wants to improve the efficiency and speed of livestock cloning as well as use cloning to preserve and multiply genetically superior animals. He envisions the day when cloning will be a fast, efficient and economically feasible breeding option for cattle, hogs and other livestock.

Compared with conventional breeding methods, clones can more accurately be endowed with specific genetic traits — for instance, tender, well-marbled meat or faster, more efficient muscle growth. Cloning also holds promise for conferring genetic resistance to livestock diseases.

“We hope to breed cattle that lack the gene or genes that make them susceptible to mad cow disease,” Stice said. “In the future we’ll be able to take cells from the very best animal and then improve upon them with genetic modifications — either knocking out deleterious genes or adding favorable genes.”

In addition to cloning farm animals, Stice wants to build on his earlier success of cloning “pharm” animals that could supply an abundance of medically important proteins.

Stice was a founder and the chief scientific officer at Advanced Cell Technology, the company that cloned the first cattle, George and Charlie, in 1997, just 11 months after Dolly made her debut. The calves opened the door not only to large-scale cloning of genetically engineered farm animals but also to the possibilities of cloning pharm animals. George and Charlie earned Stice two patents — one for cattle cloning from adult cells and one for transgenic technology, which enables genes to be transferred from one organism to another. International headlines touting the Holsteins’ arrival were short-lived. “The Monica Lewinski story broke two days later,” Stice said with a wry smile.

On the medical front, Stice’s UGA research is breaking new ground in the realm of human embryonic stem cells. These cells hold the potential to differentiate into all the various tissues in the human body. Growing specific tissues from these master cells could pave the way for a host of new medical treatments, such as nerve cells for Parkinson’s disease, and bioengineered materials that meld human cells with artificial platforms for such things as cardiovascular bypass surgery.

Since joining UGA’s faculty, Stice has helped pioneer culture techniques for growing both primate and human embryonic stem cells for medical applications. In collaboration with the Athens-based biotech firm BresaGen Inc., the researchers are among a handful worldwide that received NIH approval to grow human embryonic stem cells for federally funded research. The UGA-BresaGen collaboration holds four of the 78 approved cell lines eligible for NIH research funds following President Bush’s August 2001 decision to permit federal money to support limited research in this area.

Tangible Results

Stice, a modest and courteous Minnesota native, has a driving passion to do research that can make a tangible difference in people’s lives.

“I want to see my work result in something concrete and applicable,” he said.

By just about any measure, it already has. In addition to his many patents and laboratory successes, Stice is helping prepare the next generation of scientists who will build on his work. He lectures graduate level courses; advises master’s and doctoral students and post-doctoral fellows; and directs a vigorous UGA-based research program.

“One of the things I like the most about academe is training graduate students. Being able to help shape and direct a career is very rewarding and they in turn can energize the laboratory,” he said.

Stice is one of the University of Georgia’s nine Georgia Research Alliance Eminent Scholars. The GRA, a public-private partnership, helps recruit renowned scientists to its affiliated universities and contributes facilities and equipment to research that has economic implications for the state. Biomedical research is among the top GRA priorities.

Stice allocates 51 percent of his effort to the university and splits the remainder between two privately held Athens-based biotech companies: ProLinia Inc. and BresaGen. He serves as chief scientific officer at ProLinia, which he helped found in 1998 to develop and commercialize agricultural applications of cloning research. At BresaGen, Stice is a vice president guiding research efforts to grow human embryonic stem cells. Ultimately, his purpose is to coax these stem cells to differentiate into nerve cells for treating Parkinson’s disease.

The graduate students, post-doctoral fellows, technicians and visiting scientists in his UGA lab work hand-in-hand with the research staff at both biotech companies to advance the agricultural and medical boundaries of cloning and stem cell research.

“We operate as one unit between BresaGen and UGA,” said Ian Lyons, a research group leader at BresaGen. “Acknowledgement of shared intellectual property makes it possible.”

Altogether, Stice’s extended “research family” includes 35 people: the 15 people in his UGA lab that include technicians, post-doctoral fellows, graduate students and visiting scientists; the eight staff at ProLinia, a mix of scientists, technicians and support personnel; and the 12 at BresaGen, most of whom are researchers.

At 41, Stice is already among the more seasoned pioneers in the young field of cloning. In addition to five book chapters on livestock cloning, he has published his research findings in prestigious journals such as Nature and Science and will soon be editing a book on stem cells. Stice said he has filed “somewhere between 20 and 30 patent applications.”

One of those is for a process that vaults cloning’s success rate in cattle from a single digit to a respectable 15 percent.

“While that rate is not yet good enough to make cloning commercially feasible, it moves cloning one step closer to being a viable breeding option,” said Larry Benyshek, head of the UGA animal and dairy science department in the College of Agricultural and Environmental Sciences.

Cows, cows & more cows

Cloning won’t replace sexual reproduction of livestock, but it offers a faster way to improve livestock genetics, and that could be a big plus for cattle producers and beef consumers, Benyshek said. Cloning also could improve the genetic diversity of a breed, offering more options for future generations. “A prized bull or cow has a limited lifespan, but its genes can be stored indefinitely under lab conditions,” he said.

Stice’s cloning technique — a subtle but significant tweaking of the one that spawned Dolly — increases cattle cloning efficiency three-fold. That’s a significant improvement over Ian Wilmut’s, who cloned Dolly on his 277th attempt. Until Stice’s recent improvements, most scientists were averaging a 5 percent suc-cess rate. While Stice’s technique, which succeeds in one of every seven attempts, isn’t efficient enough to put cloning in mainstream breeding options, cloning has other qualities to recommend it.

“Presently it takes six to nine years to move the best animal genetics to the marketplace,” he said. “With cloning we can have animals ready for the marketplace in two to three years and they also will be uniform quality.”

Eight frisky Angus calves — the first fruits of the UGA-ProLinia research to improve cloning efficiency — were born between February and April 2001. By fall, they were grazing in the green pastures of a Midwestern farm. The calves were cloned from adult cells of a prized cow beyond her breeding years.

Because their genetic make-up is identical to the prized cow from which they were cloned, “genetically, the calves have a higher value to the beef industry than other calves,” Stice said.

The calves are now part of a breeding herd, but earlier this year one of the original eight clones died from heart problems. “Cloned animals have more problems and we don’t know if hers was related to cloning or not,” Stice said.

The UGA-ProLinia collaboration achieved another cloning feat this past April by cloning the first calf from cells of a slaughtered cow. KC, an Angus-Hereford cross named for the kidney cells from which she was cloned, demonstrates that the cloning process can be applied to cattle after they’ve been slaughtered and their meat graded.

“From an agricultural view, it’s very important to be able to clone cells from an animal that has gone through all the testing for meat quality and tenderness,” Stice said.
KC has tremendous implications for the livestock industry, especially since she was cloned from a cow that was slaughtered according to normal meat processing protocols, said ProLinia President Mike Wanner.

“We didn’t want to disrupt the process or change the methods slaughterhouses use in processing the meat. We didn’t want to add any additional steps,” Wanner said. “This would be a natural and easy way to access the tissue.

“The intent is to wait until the meat is graded, select those with preferred grading characteristics and bring those genetics back into a breeding program,” he said.

While KC is not the first clone from a dead animal — Italian scientists cloned a wild mouflon sheep in 2000 from cells of a sheep that had died — she is “definitely the first calf cloned from a 48-hour-old carcass,” Stice said.

Her birth signals potential benefits for both producers and consumers. The cloning technique that produced KC could mean, at the retail counter, tastier, higher quality, more consistent steaks. For beef breeders, it’s a harbinger of healthier, genetically uniform herds that produce reliable cuts of meat.

The next goal is to improve the pregnancy rate, Stice said.

“We’ve been able to get the best success rates we know of for cloning cattle, but inefficiency of the cloning process is still the single, largest problem,” he said. “In the next five years we hope cloning will produce thousands of cloned cattle for agricultural production.”

At the moment, it doesn’t come cheap. “We need to bring in $20,000 per animal. At the current state of technology, it’s really paying for the research so that we can get to the next point where we can produce an animal for $5,000,” he said.

Through the UGA Research Foundation, Stice has applied to patent the tightly guarded secret technique. It is based on a chemical inhibitor that improves the survival rate of embryos during the first seven days of growth by synchronizing cell development during a critical phase of development. (See related flow chart ). Once patented, ProLinia will license the technology.

Stice is unequivocal about who owns the intellectual property from his research. “First and foremost I am a university professor and my loyalty lies with the University of Georgia,” he said. “At the same time I think there is an opportunity for professors to be entrepreneurs. They are running their own shops, basically, and they have to be thinking, ‘How am I going to get the next grant to move my research forward?’”

From having worked in industry and from having a father who is a patent attorney, Stice has seen how conflicts of interest can arise.

“You have to be very conscious of those when you start down this path,” he said. “You have to be very open about things you’re doing. It only works if it’s a win-win situation for the university, for the professor and, of course, for the company.”

Genetic preservation

A lesser-known benefit of the UGA cloning research is its potential to help scientists preserve genetic variability among livestock and endangered animals — much the same way as gardeners save legacy vegetable seeds.

Sezen Arat, a veterinarian and reproductive disease specialist, spent two years as a visiting scientist in Stice’s lab learning the cloning process and developing technology to enhance genetic engineering. She plans to use cloning to help preserve the genetic diversity of Anatolian cattle in her native Turkey. The breed is well-adapted to Turkey’s environment but it holds limited economic or scientific interest for the rest of the world. As a result, Arat sees cloning as a way to help preserve the diminishing population that one day could disappear.

“We have some original Anatolian cows that are small but they are very strong against the environment and cold weather,” said Arat, who received a World Bank fellowship to help finance her UGA work. “We could lose our own genotype of cattle. I want to have the tissue samples so we can clone these animals and again produce the same genotype or we can keep the samples and clone them later.”

While at UGA, Arat helped develop some of the efficiency that led to cloning those first eight calves. She also contributed to developing technology that uses a marker gene — one that produces a green fluorescent protein — to verify that specific altered genes were passed on to cloned offspring. The procedure is a critical step in the long-term effort to develop transgenic livestock with, for example, disease resistance.

Arat also contributed to preliminary research on developing a universal recipient egg cell, or oocyte, that could be used to clone a variety of livestock animals or endangered species. The research also will lead to a more complete knowledge of the signals that occur between the transferred genetic material and its new cell host.

“If we can understand this relationship, maybe we can use bovine oocytes as a universal recipient to clone other kinds of animals,” including endangered species, she said.

Stice would like to try his hand at cloning endangered species, but not everyone likes the idea.

“There’s quite a bit of interest in cloning to save endangered species, but I get the most resistance to the idea from conservation groups, which are concerned about losing genetic diversity,” he said. In a counter-intuitive way, cloning actually could preserve the diversity, Stice said.

“With smaller habitats you get isolated populations of animals and then they become inbred. That’s why the Florida panther may have problems reproducing,” he said. “Cloning may be useful in introducing new genetics from a different population somewhere else.”

In January 2001, scientists at Advanced Cell Technology, a company Stice co-founded in 1994 with his doctoral adviser, James Robl, cloned an endangered wild ox or gaur. Noah, the first endangered animal to be cloned, died two days after birth from an opportunistic infection.

Pig Tales

When Stice arrived at UGA in 1998, no one had yet succeeded in cloning a pig. Using the basics from his cattle cloning technique, Stice set out to do so. The project was especially challenging: Pig eggs and embryos are far more sensitive to handling than are those of cattle. Each step in the cloning process jars the fragile egg cells and reduces their viability. For reasons the team couldn’t quite pinpoint, very few of the fused eggs were surviving and growing into embryos that could be implanted into surrogate sows.

Time and again the UGA-ProLinia cloning team collected hog eggs, prepared them to be fused with genetic material from a prized hog, stimulated them to behave like fertilized eggs — and then watched and watched. More often than not, nothing happened. Weeks stretched into months with the team carefully, tediously, relentlessly extracting cell nucleus after cell nucleus, one at a time, from hundreds and hundreds of sow eggs and then ever so gently substituting nuclei from skin cells from the boar to be cloned.

Once or twice a week, someone in the lab — usually a graduate student — had to make the four-hour round-trip trek to the slaughterhouse and replenish the supply of sow eggs salvaged from the ovaries of hogs destined for ham, sausage and bacon. But no amount of babying the delicate cloned embryos produced the results they had gotten with cows.

“In cloning, everything has to come together and everything has to be right,” Stice said. “We’re doing something different than everybody else in our effort to clone adult animals in a more efficient way. We’re trying to use resources and techniques that other people haven’t.”

This past May, their arduous efforts finally paid off when three healthy, pink pigs were born.

Although it’s more difficult to get pig embryos to grow and thrive in the first seven days before they are placed in a surrogate sow, once the sows become pregnant, matters seem to get easier. Stice said he believes that the hog industry actually will move cloning into mainstream commercial operations quicker than cattle producers will.

“In five years I think you’ll see the number of cattle being produced from clones will be in the hundreds of thousands,” he said. “And it will probably be even sooner for cloned pigs because the industry is set up to adopt the technology more easily.”

Better cloning efficiency will mean lower pork production costs and quicker response to consumer demands, he said.

“For example, the Japanese like darker pork meat with more fat. We’ve bred our animals away from these qualities,” Stice said. “Cloning makes it much faster to respond.”

A report issued by the National Academy of Sciences in August, which concludes that cloned animals don’t seem to pose risks for human consumption, may speed cloned pork chops and Porterhouse steaks to the butcher’s meat case.

As a result of that report, Stice said he “expects the offspring of cloned meat will be approved for human consumption.”

Stice also sees his hog cloning research providing benefits beyond the dinner table. He plans to branch out toward medical applications, not to clone spare parts for people, but to develop animals that can serve “as models for research into diseases such as Alzheimer’s.”

“I’d like to use cloning techniques to develop an animal model for Alzheimer’s,” he said. “The mouse is not a good model. A mini swine could be a test model for human Alzheimer’s.”

Stice sees the mini swine as a way to speed the development of more effective drugs and treatments for Alzheimer’s and for another disease that strikes people at the opposite end of the age spectrum. Ataxia Telangiectasia, a progressive, genetic disease caused by a mutation in just one gene, begins with loss of balance among small children and results in their death, often by the late teen years.

“We’re looking at knocking out that gene in a mini pig so it could be used as a model for AT,” he said.

The war against disease

Animal cloning is a natural complement to Stice’s other research: nudging embryonic stem cells down specific developmental pathways for medical applications.

The microscopic cells derive their great therapeutic potential from an almost magical ability to transform into all 220 or so types of the body’s tissues and cells.

Together, UGA and BresaGen are helping unravel mysteries of getting stem cells to produce specific cell types.

“Once you get the stem cells growing, they’re easy to maintain,” said John Calhoun, a doctoral student who helped develop stem cell culture techniques.

With some routine “gardening” a culture will grow and grow and grow, almost in perpetuity, he said. The hard part is getting them to develop into nerve, muscle or artery cells, for instance.

Such knowledge is a first step in developing replacement therapies for diseases involving loss of particular cells, such as nerve cells in Parkinson’s disease. It also holds promise for engineering surgical replacement parts.

“We’re not looking at developing an organ,” Calhoun said. “We’re learning about the signals that stop and start the differentiation that occurs during development.”

Long-term, Stice wants to develop specific nerve cells, called neural stem cells, for alleviating Parkinson’s symptoms.

When nerve cells in certain brain regions of a Parkinson’s patient begin degenerating and dying, they stop producing dopamine, a signal transmitter critical in relaying nerve messages within the brain. Once 60 to 80 percent of the dopamine-producing cells have died, a patient begins to show the characteristic tremors, slowed movement and other symptoms of this incurable and eventually fatal disease.

Stice collaborates with researchers at other universities who already have shown that implanting nerve stem cells into specific brain regions of a Parkinson’s patient may reduce symptoms and restore functions lost to nerve cell degeneration.

“One of our collaborator’s patients was able to run out of the World Trade Center on September 11th and get to a train station,” Calhoun said.

Stice knows there’s still much work ahead. “We’re about two years away from human trials with the Parkinson’s research,” he said.

In the meantime, the UGA and BresaGen teams are pushing ahead on animal model studies for the disease and making uniform, functional dopamine-producing cells that mimic those in the human body.

“There’s a lot in making nerve cells that goes beyond dopamine,” Stice said. “We need to do functional tests — does the cell in a petri dish function like a dopamine-producing cell in the brain?”

Nobody knows yet. But the UGA-BresaGen collaborators are finding out by using a special rat model lacking dopamine-producing cells in specific areas of the brain. Rat behavior undergoes pronounced changes when neuron stem cells are introduced into specific brain regions.

“We know the process works and the test is reliable. It’s a long-term study and will take time to pinpoint the specific cell development conditions to obtain appropriate renewed brain function,” said Clifton Baile, another GRA Eminent Scholar at the university who oversees the behavioral tests. Baile, who is ProLinia’s CEO and board chairman, played a key role in recruiting Stice to UGA.

“If we found a cell type to cure Parkinson’s in rat, the next step would be to induce Parkinson’s-like symptoms in primates and look for the same kinds of effects,” said Ian Lyons, who directs BresaGen’s part of the stem cell research. “It’s an exciting part of biology to be in.”

And so is Stice’s other stem cell research with Robert Nerem’s team at the Georgia Institute of Technology, which focuses on cardiovascular applications of stem cells. In this project, Stice is developing endothelial cells from embryonic stem cells and also working on ways to bypass rejection issues.

Endothelial cells line the blood vessels. The Ga. Tech-UGA research group wants to use those cells to engineer tissue for such applications as cardiac bypass surgery or other vascular replacements, he said.

Of cloning and stem cell research programs, Stice sees the latter as having greater healthcare applications.

“It can be used in so many different ways to benefit people and that will be very important,” he said.

But for all his success so far, Stice said he believes the most important discovery of his career “is yet to come.”

“I hope I can help a student make a basic discovery,” he said, “on how cells determine what to become — for example, a nerve cell instead of a muscle cell — thus benefiting both animal cloning and cell therapies.”

For more information, access Stice’s Web site http://www.uga.edu/caes/biotech/stice.html, ProLinia’s Website www.prolinia.com, or BresaGen’s Web site www.bresagen.com

Judy Bolyard Purdy is the University of Georgia’s director of research communications. She has degrees in biology, botany and journalism and has won several regional and national awards for writing.

Editor’s Note: The Learning Channel will feature Dr. Stice’s work in its upcoming program, Ten Ultimate Technological Inventions of the 1900s.

THE UNIVERSITY OF GEORGIA RESEARCH MAGAZINE : www.researchmagazine.uga.edu