Jerry Grillo, September 2010Two UGA scientists make a remarkable breakthrough
Reproduction can be pressing business, fraught with challenges. But two University of Georgia scientists made a breakthrough discovery in reproduction and regeneration that has thrown open the doors to wide-ranging possibilities, including new therapies for devastating human diseases and the preservation of endangered animal species.
Steve Stice and Franklin West won what amounted to a hotly contested race to become the first scientists to produce induced pluripotent stem (iPS) cells from adult livestock.
Like human embryonic stem cells, iPS cells can turn into any type of cell in the body. But, while embryonic stem cell research has been attacked and condemned by religious conservatives and lawmakers trying to curry favor with them, iPS research is seen as a less controversial approach, and the discoveries at UGA represent a promising new chapter in the field.
“This is very significant, very promising. All of the stars had to be perfectly aligned for this to work right,” says Stice, a Georgia Research Alliance Eminent Scholar in the UGA College of Agriculture and Environmental Sciences and director of UGA’s Regenerative Bioscience Center.
“We had quite a few competitors working toward the same goal. That’s why it took some time to get to publication. We all review each other’s papers, often becoming highly critical,” Stice says. “So, we had some competitors, but we got there first.”
Stice’s team injected human genes into pig bone marrow cells, reprogramming them into pluripotent stem cells, which were then injected into pig embyros, which were inserted into mother pigs. The piglets – technically they are chimera, because they also contain human genetic factors – were born last September.
The new technology developed by the two UGA researchers will basically allow scientists to make custom-designed pigs that will serve as a better research model for human disease and as a source of cells and organs for regenerative medicine, according to Stice, who says this new research tool may be used to determine which stem cells – adult, embryonic or induced pluripotent stem cells – work best for specific diseases.
The discovery could pay fairly quick dividends for a research project under way with Emory University scientists, who are researching better therapies for diabetes. And Zoo Atlanta is working with Dr. West in the area of endangered species reproduction and conservation. Meanwhile, the scientists have secured a major grant to support studies that they hope will lead the way to healthier, disease-resistant, environmentally friendlier livestock that could help reduce hunger and poverty in developing countries.
“This is still early, and we need to determine whether we can get these stem cells to form more than chimeric animals,” Stice says. “Can we get them to produce additional offspring through traditional and other techniques? That’s something we need to do on a very basic level.”
The piglets born last September are now breeding, and Stice expects to find out this fall whether they reproduce.
Franklin West often keeps mad scientist hours, and this was one of those nights that he couldn’t sleep. So he and a grad student, Jenny Mumaw, were running gels in the bleary hours, trying to track whether or not the induced pluripotent stem cells they had developed through reprogramming the bone marrow cells were contributing to the piglets.
“We were doing the analysis to show that our pigs were truly chimeric,” West says. “A lot of people have developed cells that look, talk and walk like stem cells, but to actually know they contributed to a developing animal was simply amazing.”
One way to do the analysis is to follow the gene that reprogrammed the original bone marrow cells into iPS cells. The scientists had designed probes to signal when the gene was present in the DNA of the developing piglets.
“I never know when I’m going to get an email from Frank,” says Stice. “He eats, sleeps and breathes this stuff. He’s up at all hours thinking about this stuff.”
West says it was three or four in the morning when he and Mumaw saw the first bands indicating the human gene was present in the fetal pigs.
“Our probes lit up,” West says, describing the white bands appearing against a black background. “We were surprised and excited.”
Stice says, “We really didn’t believe it at first. Frank had to do it several times to convince us.”
That time in the lab was the second big “A-Ha” moment for West. The first was after the scientists had reprogrammed the pig bone marrow cells using the human factors.
“I was like, what are the chances they’re actually going to reprogram the pig cells, because that’s a bit of a leap still, and for it to have worked was, first of all, kind of shocking and exciting to me,” West says.
“When we looked under the microscope and saw that they were expressing stem cell markers, and they looked like stem cells, that was a key thing. Because you can make a cell express all kinds of things, but to look and act like stem cells, that was kind of crazy.”
Scientists had successfully used iPS cell technology in mice, but the mouse isn’t always an acceptable model to study human disease, and mice are definitely not a good source of tissue and organs for therapy. Pigs, however, are biologically and physiologically comparable to humans and prone to some of the same health problems.
Stice, who has been trying to solve the riddle for 20 years, credits his young research assistant West with finally perfecting the method.
“It was Frank’s perseverance that made the difference,” Stice says. “His efforts were the key ingredient.”
West came to biotechnology in a roundabout way. A graduate of Morehouse, where he studied biology, he wanted to pursue a career in ecology. He studied intertidal snails and went to Kenya to study yellow baboons.
“A phenomenal experience overall, but at the end of the day I thought I could help more species from a biotech approach,” he says. “That’s why I teamed up with Steve, because at the time he was really interested in helping endangered species through cloning.”
Around the time West entered the Stice lab was around the time Stice stopped trying to clone animals for the purposes of animal conservation.
“We didn’t get a very positive reaction from the community,” Stice explains.
The induced pluripotent stem cell process circumvents the more problematic and controversial cloning process and makes it easier to develop the genetic changes necessary to develop pigs as an optimum source of cells and organs for transplantation. It also avoids, sort of, the use of human embryonic stem cells – another contentious issue, especially on political grounds.
“We built on what we’ve learned from our research in human embryonic stem cells over the last six, eight years,” Stice says. “That’s an important point, because this newer technology is still a work in progress.
“Will these iPS cells make good sperm cells? Will they make good egg cells? Will they make good islet cells for making insulin? Those are all important questions that we don’t know the answers to. Whereas, we know that the embryonic stem cells have the capability of doing certain things other stem cells can’t.
“To say, ‘Now that we have iPS cells we don’t need to work in embryonic stem cells’ is just dead wrong.”
Stice doesn’t want to close the book on any stem cell research.
“Right now we don’t know where the cure for Alzheimer’s or Parkinson’s is going to come from. But let’s say the cure is behind one of three doors,” he says, borrowing an analogy from Dr. Story Landis, director of the National Institute of Neurological Disorders and Stroke.
“Behind one door are embryonic stem cells, behind another are adult stem cells, and iPS cells are behind the third door. Wouldn’t you want to look behind all three doors?”
New Tool for Research
In Greek mythology, a chimera was a nightmarish creature with the head and body of a lion, a tail that ended in a serpent’s head, and jutting out from the spine, a fire-breathing goat’s head.
“‘Chimera’ is probably not the best word. It has an unfortunate connotation,” Stice says.
In biology, a chimera is an animal or plant made up of genetically distinct cells from two or more zygotes, which may be of different species. Before now, chimerism has typically been created in mice by researchers as a way to better evaluate human cells, considered a critical practice in translating scientific discoveries into therapeutic medicine. Again, pigs are considered a better model for such evaluation, and the new process developed by Stice and West should be a valuable tool for a project under way in partnership with Emory scientists who are researching better therapies for diabetes.
Islet cells from pigs could be the major breakthrough for the treatment of Type I (juvenile) diabetes, but they ultimately are rejected by the human immune system, requiring additional therapeutics.
At Emory, Colin Weber and Susan Safley have been working diligently on developing better therapies for diabetes and have collaborated with Stice for some of their research. But the researchers have not yet embraced the recent breakthrough of the UGA scientists.
“Pig islets, if you can get them to work, could be a universal soldier,” Weber says. “But treating human diabetes is still a moving target, what I call a noble goal, something worth getting up in the morning to do. And there’s always a new idea around the corner.”
The idea, says Stice, is to develop pig islet cells (that produce insulin) to replace the pancreatic islet cells that aren’t up to the job in people with diabetes, making genetic modifications in the pig cells to diminish and possibly eliminate rejection.
“Insulin from pigs has been used a long time, and it does well, so if we can get around the rejection issue, that’s something that makes sense from the get go,” Stice says.
Meanwhile, West – who now has a lab of his own on the third floor of the Edgar L. Rhodes Center on the UGA campus, one floor above Stice’s lab – is focusing his research on germ cell development. Germ cells lead to the production of gametes (egg and sperm cells). His research in stem cells and animal reproduction may have implications for hundreds of human diseases and problems, but the immediate outside interest has been focused on what it could mean for endangered animal species.
He’s collected cells of two endangered species – the clouded leopard and Sumatran tiger – from Zoo Atlanta, with hopes of creating iPS cells that will ultimately morph into sperm cells.
“This technology is a potentially powerful tool,” says Dwight Lawson, senior vice president of collections, education and conservation at Zoo Atlanta. “We think it can be perfected to the point that it would allow us to go back and collect biological samples of animals that have been dead for years, [and] turn those samples into germ cells that can be used. We’d be rediscovering genetic diversity that we didn’t have access to before.”
West’s thought is to create a cell bank.
“If you have an endangered species, and there are only 50 males left, you can isolate cells from them and create a bank of cells that you can turn into sperm,” he says. “Some species have reproductive problems or they don’t breed well in captivity, and often their last refuge for survival are zoos and wildlife parks and sanctuaries.”
For Lawson, using the new iPS technology developed by Stice and West for the preservation of animal species is, “a no-brainer, the kind of thing we try to do in the course of every week. Our role is to provide biological samples and see if we can get this technology to work for some of these rare and endangered species”
The iPS technology and discoveries emerging out of UGA create the likelihood of making stem cells from just about any species, says Stice, whose goal is to use the research to battle hunger and poverty in developing nations through the development of heartier livestock.
“For example, you can start to select the genetic background that has the best disease resistance through this technology,” Stice says.
One deep-pocketed foundation with a global reach has just committed some funding – “could be a million dollars or more,” Stice says – for a new project that would involve using the iPS technology for chickens, a project designed to be of value to developing countries that need disease-resistant livestock.
Stice says the foundation prefers not to be mentioned as a supporter of the research until the scientists get further into the project.
The subjects of stem cell research and chimerism provoke passions on either side of a national debate centered on what is or isn’t ethical. One of the reasons Stice, West and other researchers are focusing so much on iPS cells is because of the inherent controversy over human embryonic stem cell research.
“Because we’re always in this state of not knowing whether we can work with this stem cell line or this other stem cell line,” Stice says, “as researchers, we always have in the back of our minds, ‘Well, we’re doing all this work with this particular line. Are we gonna be able to use it in the next administration that comes along?’
“It’s politically charged, and politics always gets in the way, and no matter what side you’re on, the issue always affects you.”
So there is a sense that iPS cell research does not stimulate the same ethical concerns that surround and hound human embryonic stem cell research. But neither Stice nor West believes their iPS technology will replace embryonic stem cell research and its potential to alleviate human pain and suffering.
“We have this new technology, and it’s exciting. It’s got great potential,” West says. “But it’s only a tool, and it does not tell the complete story by itself.”
Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.