BY STEVE LUNDEBERG
More than 40 years ago, a television astronaut-turned-spy named Steve Austin rocketed into the pop culture stratosphere and blasted the concept of replacement body parts into the public consciousness.
Austin, as the story went, had been badly maimed in a horrific aircraft crash.
He was subsequently outfitted with four human-built replacement components: two legs, one arm and one eye, each more highly functioning than the original.
For Austin, hero of The Six Million Dollar Man sci-fi drama, the medical cure came via bionics – mechanical systems engineered to work seamlessly with his natural tissue.
Four-plus decades later, the dream of lab-grown limbs and organs for those who’ve lost them to injury or illness is closer than ever to reality. Oregon State University scientists led by molecular biologist Chrissa Kioussi, from the College of Pharmacy, are at the forefront of the charge.
Kioussi, computational systems biologist Stephen Ramsey and pharmacologist Theresa Filtz have learned that Pax3+ cells – precursor cells for skeletal limb muscles – also give rise to neurons, blood vessels, blood cells, bone cells and immune cells. This is a key step toward generating replacement components. Ramsey is an assistant professor at OSU’s College of Engineering and Filtz is an associate professor at OSU’s College of Pharmacy.
“We’re working with differentiated cells that are no longer dividing, but we can put them in a Petri dish and differentiate them and force them to become something else,” says Kioussi. “It’s a combination of developmental biology, genetics and bioinformatics.”
By labeling cells based on expression profiling of sequence-specific transcription factors, the researchers can give a molecular code to each cell, making it distinct in time and space. Transcription factors turn genes on and off to make sure they’re making the right products, usually proteins, in the correct cell at the correct time and in the correct amount – for the entire life of the cell and the organism.
“During the progression of a cell, the molecular code changes,” says Kioussi. “The labeling and coding are beautiful because we can use this information with the potential to fix so many genetic or environmentally caused disorders. This gives us the possibility to make an entire cell lineage, and therefore an entire organ, in a Petri dish.”
Building an Arm and a Leg
Using a mouse embryo model, the researchers compared gene expression profiles of the Pax3+ cell population over several days and identified genes involved in the skeletal, muscular, vascular, nervous and immune systems. “All of which go into making a functional limb,” adds Kioussi. “Expression of genes related to the immune, skeletal and vascular systems showed big increases over time, which suggests Pax3+ cells give rise to more than muscles – they’re involved in patterning and the three-dimensional formation of the forelimb through multiple systems.”
The research opens the potential to use stem cells to grow a new arm or leg or organ for someone who’s lost a body part to disease or an accident – like the one that led to Steve Austin becoming The Six Million Dollar Man.
“That cell population can give rise to so many different cell types; we can isolate cell populations and upon creating the perfect environment for them, they become something else over time,” Kioussi says. “It’s a great source we have to fix different organs with issues from injury, toxicity from drugs, anything that can generate cell death in any part of our body.”
She likens the research to a mechanic disassembling an engine down to every last nut, bolt and washer, which calls to mind wiring and other components that would become exposed whenever Austin suffered an injury to one of his replacement limbs.
“An engine has so many little parts, and you can’t fix a broken engine if you don’t know what all of these little parts do,” she says. “Likewise, you can’t make a limb if you don’t know about all of the cell parts within the limb. You can’t use only bone or only muscle or only veins – you need everything working together.”
The complexity is mind-blowing. For example, humans have about 20,000 genes – 2,000 of which are transcription factors, which regulate other genes. “Those 2,000 are the machinery responsible for the function of the other 18,000 genes,” Kioussi notes. “Within those 2,000 genes are a small population called homeobox genes, and of these, 200 are expressed very early in development. These are the patterning genes, determining what organs are going to be formed, how they’re going to be positioned in-body. They’re the genes that tell you you’ll have two eyes, one nose, the correct number of teeth, the correct number of digits, etc.”
From those 200 genes, the researchers have identified a handful, which play a very specific role in the formation of muscle tissue, a process known as myogenesis. Muscle fibers are formed when myoblasts – progenitor cells at a stage between stem cells and mature, functional cells – fuse together.
“In the lab, we can eliminate the function of a gene involved in skeletal muscle during specific developmental times to get an idea of what the function of that gene is in space and time,” says Kioussi. “That lets us learn what is necessary for this myoblast to become a myocyte and then a myofiber and then a functional muscle.”
Breaking down, sorting out and understanding the dizzying amounts of data generated from measuring the expression of all genes in cells at various time points is where the computational biologists come in.
“It’s like breaking a story or book apart,” explains Kioussi. “You randomize it, dissect it, then you’ve got this random assortment of letters. The computer scientists take the random assortment and use algorithms [software that identifies patterns] to try to make sentences out of them. Then they take the sentences to the biologists, and we try to put this together and make a chapter and
then a story.”
Systems biology is by necessity a team endeavor, Ramsey adds, since massively parallel experimental methods are used hand-in-hand with sophisticated computational tools.
“Organ growth involves the coordinated regulation of expression of thousands of genes,” he says. “It’s like a gene regulatory circuit diagram in which genes turn other genes on and off at precise times and in precise anatomic locations during development. The diagram is enormously complex and largely undiscovered; we only directly see the developmental program’s grand finale, a working organ or, in the case of disease, a dysfunctional one.”
But with careful experimental design and execution, the huge amount of data from cells and tissues can be analyzed to infer the circuit diagram of the genes that are regulated by a specific protein such as Pax3. That protein gives rise to the Pax3+ precursor cells that Kioussi, Filtz and Ramsey use in their experiments.
“We can determine if two gene networks are connected by having very similar expression patterns,” says Ramsey. “We then compare the networks under two different conditions – for example, under normal development, and also with a key regulatory protein disabled.”
Think of it as modifying the circuitry in one of Steve Austin’s bionic legs and then seeing how it functions relative to the other leg.
“The differences between those gene networks, combined with other types of ultracomprehensive cellular measurements, enable researchers to explain part of a developmental gene regulatory program,” notes Ramsey.
Ethical Questions
Unlike some branches of regenerative medicine research, the course being pursued by the labs of Kioussi, Ramsey and Filtz has not encountered any ethical battles, adds Kioussi.
“We’re not using human embryonic stem cells,” she says “We are working with mice that share 92% of our human genes to repeat organ development. Our type of work is very well embraced within the scientific community for the good it can bring to humanity.”
While no protesters are picketing outside Kioussi’s lab, the research necessarily brings up a few points to ponder, says Courtney Campbell, a medical ethicist in OSU’s College of Liberal Arts.
“These sophisticated technologies are going to be expensive,” he notes. “So how much priority should they receive given that for 27 million people in the U.S., where there’s no universal access to health care, the only access to medical care is the emergency room? Is there a way to make sure the benefits of the research are available to all regardless of ability to pay?”
Another aspect to consider, according to Campbell, is to what extent the mouse model is a good one – what are the chances that humans, a more complex organism, might be subject to procedures that prove problematic?
“Which raises the question of informed consent,” he says. “Will individuals in the first trials have a robust sense of the risks they might experience?
“Growing limbs and organs would obviously be an enormous benefit for people who suffer from various afflictions,” adds Campbell. “But are there other ways to provide benefit more quickly? Robotics provide some function, but they’re pretty crude, so this regenerative approach sounds really good by comparison. Ultimately, is it going to be better to go this route or another one, such as via a person’s own stem cell regenerative processes? We don’t know. At this point, though, we have to allow the research to go forward.”
Kioussi, meanwhile, believes in the research enough to state, emphatically, that lab-grown organs derived from a person’s own cells will become commonplace during her lifetime.
“I can provide cells that are programmed to deliver the information needed in an organ. I can figure that out with the computational biologists,” she says. “Now we need to recruit the engineers with their 3-D printing to build the scaffold of a limb or of a muscle group or for an organ, and then I can grow my cells into that. We generate building blocks in the lab, and the bioinformation tells us where the block can be placed – in the foundation or in the roof.”
Which means real-life science is poised to race past the technology behind The Six Million Dollar Man with even greater speed than Austin’s bionic legs afforded him.
“This is not fiction anymore – it’s reality,” says Kioussi. “It’s part of our movement toward precision medicine. We’ll get to a point where everyone has their genome information in the palm of their hand, on their phone. You’ll go to the hospital and everyone will know what your condition is, or what your genome is, what antibiotic to use, which anesthesia. It’s all part of the new approach we’re using to better understand human disease and treat the patient.”
