In a typical September morning at the Kennedy Space Center in Florida, the humidity is high and rising. Researchers Timothy Hammond and Thomas Goodwin stare out the bank of windows in the Operations and Checkout building, doing their best to hide their anxiety. Some 8 miles ahead, the space shuttle Atlantis is on the pad, waiting for word from Houston that the launch can proceed.
On September 8, 2000, Atlantis blasted into space for mission STS-106, a two-week flight. One experiment on board proved that some genes respond vastly differently in space than in culture conditions on Earth.
Photograph courtesy of NASA
This launch is the second-to-last mission to the new International Space Station before the first full-time residents move in. The astronauts and cosmonauts plan to deliver supplies to the service module, install a toilet, and complete some electrical work. Media reports focus on the astronauts, but the duo at the windows is worrying about the well-being of another group of passengers.
During the last three weeks, Hammond, Goodwin, and a team of five biologists have painstakingly nurtured more than half a billion human kidney cells in petri dishes, injected them into a liquid that looks a lot like cherry Kool-Aid, and divided them into 36 sandwich-sized bags. Now four of those little plastic bags, holding some 58 million cells, are nestled in a container the size of a small coffee can within a locker on the mid-deck. Once the shuttle enters the microgravity of space, they expect the cells will grow.
The experiment may seem a bit mundane, but it's far from it. Researchers have known for some time that cells respond differently in different environments, say, in a petri dish as opposed to a human body. What they haven't known, and what Hammond and Goodwin hope to validate, is that cells grow remarkably well in microgravity. "If you take a kidney or liver or brain cell out of the human body and put it in a standard, flat culture on the ground, each will lose its special features, meaning, essentially, that they no longer 'know' they are kidney, liver, or brain cells," says Hammond, a kidney specialist at Tulane University Health Sciences Center/Veterans Administration, who also heads up the center's program on environmental astrobiology. "That means the genes no longer produce or 'express' the distinctive proteins of a kidney, liver, or brain. They lose the ability, in other words, to regulate their genes and remain differentiated. But in space, cells become happy, and they grow."
The potential of cell growth in a weightless environment is nothing short of extraordinary. "We want to grow these cells in three dimensions, more like they would grow in the human body, which they seem to do pretty well in the microgravity of space," says Goodwin, manager of biotechnology flight definition at NASA's Johnson Space Center. "Then we want to dissect the elements of the genes to find out what proteins they're producing. What we're doing is molecular genetics in space, which is, in a manner of speaking, a lot like taking a clock apart to see what makes it tick."
Researchers hope to move beyond the mere mapping of the 30,000 to 40,000 genes in a human and move inside the genome itself to figure out exactly how genes do what they do and why. Understanding the cellular events that "turn on" specific genes will open a new frontier in biology and medicine: new systems for culturing cells and growing organs, as well as new diagnostic and screening systems. It could lead to ways to regenerate tissue in space and eventually create replacement organs from a person's own cells.
For Goodwin and Hammond, the wait for a significant test in space has been six years. Still, their progress has been so promising that they managed to get a coveted slot on mission STS-106. If Atlantis doesn't fly today, they might have to wait another year to get rescheduled.
At T minus nine and counting, however, the clouds dissipate, and at 8:45 a.m., right on schedule, the solid rocket boosters rumble. Atlantis roars into the sky. Approximately eight and a half minutes later, the cells are free floating in space. The journey has begun.
For more than a century, biologists have studied cells using a variety of culturing techniques. But in the mid-1980s, as NASA explored the idea of sending humans into space for months instead of days, no one knew how long-term stints in space might affect the human body. So the space agency assembled a design team at Johnson Space Center to find a way for NASA biologists to study what happens to human tissue in modeled microgravity conditions.
Kidney cells grow microvilli— hairlike fibers— that help absorb water and nutrients. Rat kidney cells grown on a 21-day flight on Mir (left) show more microvilli than cells grown in cultures on the ground (right).Photographs courtesy of Timothy Hammond
Physician and electrical engineer David Wolf and engineers Charles Anderson, Ray Schwarz, and Tinh Trinh created a device dubbed the Bioreactor that simulates some of the conditions cell cultures experience in microgravity. Rotating the Bioreactor on its side between 10 and 30 revolutions per minute simulates free fall, the same condition in which the space shuttle orbits Earth. When the shuttle achieves orbital velocity— 17,500 miles per hour— the effect of gravity is reduced up to 10,000-fold, creating an environment of weightlessness.
In June 1987, Goodwin began to conduct the first complex biological experiments with the Bioreactor: "We began creating cell-culture models that we had never been able to create in ground cultures before, three-dimensional tissues that looked like, and in some cases functioned like, tissues in the human body."
Hammond heard Goodwin present his results in 1990 and proposed the next step: Fly the cells into space, in real microgravity. The two embarked on a collaboration that suited their talents and temperaments: Hammond, an ebullient, established physician and researcher with an eye for fostering the practical applications of this work; Goodwin, a young visionary working his way toward the first Ph.D. in space physiology and engineering science. Within a few years, they had persuaded NASA to fly their first preliminary experiment aboard the Mir space station. Goodwin's Bioreactor colleague David Wolf, who had gone on to become an astronaut, carried six bags of rat kidney cells inside a NASA-developed incubator onto the Russian space station during his visit from September 1997 to January 1998. The cells responded and grew, Wolf says, forming delicate structures of tissue that were vastly different from conventional cultures grown on the ground and different from those grown in the Bioreactor. "The tissue was organizing, and it happened fast, starting in the shuttle on the way up." This phenomenon, Goodwin explains, "was a result of differences in cell-to-cell signaling and the formation of tissuelike scaffolding, just like one would see in tissue inside the human body."
Word rippled through various divisions of NASA, and Hammond and Goodwin got a slot for their first flight of human kidney cells on the Neurolab mission, STS-90, on the space shuttle Columbia in April 1998. The results were dramatic. "We were stunned by the number and magnitude of the changes measured after six days in space," Hammond remembers.
The next step was to figure out what happened to the genes and when. That's what the experiment aboard the Atlantis was designed to study: how the cells' genetic activity would change. In order to identify results specific to microgravity, the team has also conducted parallel ground-based studies. These included controls for gravity and vibration (which mimicked the exact profiles of the shuttle launch), as well as one in the Bioreactor, and of course, a standard ground control. "Vibration, for one, is a potent mechanical force, and that is what this experiment is all about, finding out how the space environment impacts genetic expression, which other mechanical culture conditions might also impact expression, and how they do it," says Hammond.
On September 20, Atlantis returned to Earth. Hammond picked up the bags of kidney cells and hand-carried them back to his lab at Tulane. There the cells were divided up and run through the same five analyses as those in the control group, which had remained on the ground. The Medical College of Georgia ran an independent analysis to double-check the results.
One of the several cylinders (left) on board the space shuttle carried bags containing human kidney cells (right). After two hours in flight, the cylinder automatically injected a substance into the bags that preserved the cells' RNA for later analysis.Photographs by Jonathan Kantor
As anticipated, microgravity produced more change than any other culture condition. More than 1,600 genes showed three times as much activity as genes in cells cultured on the ground. Not only did the results validate their first flight experiment, but they blazed a new trail for scientific experimentation. "Gravity has never been considered a variable in mainstream scientific research, because gravity reduction is not something scientists have ever been able to modulate," says Goodwin. "Soon we'll be able to modulate the gravity environment to elicit the kinds of responses we want."
"The research cannot yet tell us directly about how the astronauts on long-term space voyages will be specifically affected," says Hammond. But he and Goodwin are certain that the effects will now be studied with more interest.
At the moment, the two researchers are focusing on pinpointing changes in individual cells two hours into the flight. "We saw distinctive patterns of change in the different culture conditions," says Hammond. "One group changed only in space, another both in space and in the Bioreactor, and another group of genes changed in all the cultures." Hammond thinks they'll eventually find that different culture conditions will be optimal for different genes or gene groups. Some genes, he suggests, may respond better in space, while others may respond better in the Bioreactor, or what is generically called modeled microgravity.
The genes that showed the most activity proved to be those involved with fundamental life processes, such as driving cells to maturity or prompting programmed cell death, the body's way of getting rid of worn-out cells. "More than 1,600 genes changing in microgravity is a lot, and these are some of the most important genes in the body," says Goodwin. The single gene that showed the most increased activity encodes gamma interferon, an immune-system activator. Gamma interferon is a powerful— and at the moment, expensive— antiviral used for treating HIV; hepatitis C, D, and E; and cancer. Kidney cells in space made 15 to 20 times as much gamma interferon as they do in cultures on Earth.
Hammond and Goodwin also looked at the production of two critical hormones— erythropoietin, a hormone that promotes the growth of blood cells, and vitamin D3, which helps build strong bones. Because chemotherapy, some antiviral drugs, and kidney failure destroy the cells that produce these hormones, erythropoietin and vitamin D3 are valuable commercial products, with annual sales totaling several billion dollars. The kidney cells made five times more of these hormones than they do in Earth-bound systems.
There is much left to untangle in the data. For example, the team needs to tease apart which components activate the other genes under study. Making a particular molecule involves complex interactions between a gene or genes and the control proteins that initiate the genetic activity that assembles that molecule. "It's just like a child's matching game," Hammond says. "We now know many of the proteins that go into the nucleus to turn on genes or gene clusters, and we know which genes are turned on. What we're still looking at with many of these genes is which control proteins turn on which genes or gene clusters." And, he adds, they're ready for surprises. "We really have no idea of all the things we may find."
The goal, of course, is to figure out the particular cues that trigger the manufacture of specific, desired molecules, then use that knowledge to make gamma interferon, erythropoietin, vitamin D3, and the like more easily on Earth in the Bioreactor. "We're not flying to make a factory up there— not right now anyway," says Hammond. "We're flying to bring the knowledge back here to use on Earth."
Hammond believes such analyses will yield a host of new tools for medicine, including a better method for testing drugs. Drugs are screened in two-dimensional culture systems or in animal models, which have inherent difficulties because of differences between animal and human physiology. To get better results, he says, scientists need a human cell line that expresses the six major enzymes in the kidney and liver that metabolize drugs: "That kind of cell line is not available currently, but by learning very specifically which proteins turn on the drug-metabolizing enzymes in space, we will be able to turn on those proteins down on Earth and develop a much better commercial system to look at drug interactions." A more effective drug screen, he adds, "could help millions of people, because drug interactions and adverse drug reactions are the number one reason for prolonged hospital stays."
Some of these new applications are already in development at StelSys, a Baltimore-based biotech company. Last September StelSys obtained a five-year licensing agreement with NASA for exclusive rights to specific fields of use on 13 NASA patents, including the Bioreactor and various processes related to Hammond and Goodwin's work. One of their projects uses modeled microgravity to create three-dimensional tissue cultures of liver and kidney cells. They're using these cultures to develop better ways to test how a drug is metabolized.
StelSys is also working on a device for detoxifying blood for patients with damaged livers. "Most of the liver-assist devices currently in operation use liver cells, either human or pig. But the difficulty lies in keeping these cells behaving like liver cells for a sufficient period of time, because they lose their ability to stay differentiated, or to know that they are liver cells," says Paul Silber, president of StelSys. "What's remarkable about this microgravity Bioreactor is that it helps preserve long-term differentiated function of the liver cells. That's something that no one else has really been able to crack or figure out how to do in 20 years of working with liver cells. It's just a quantum leap."
Hammond and Goodwin are building the experiment for another flight, slated for next May. They hope to study in detail the processes that turn on genes to produce vitamin D3.
The cells grown in space (left) have green concentrated in the nucleus, while cells grown on the ground (right) don't. The green indicates that a protein (the vitamin D receptor) has moved from the cell's surface to turn on genes in the cell's nucleusPhotograph courtesy of Timothy Hammond
While Hammond prefers to emphasize the projects that will yield the most immediate results and do "the most good first," Goodwin and others look further ahead. "This research heralds new possibilities for space research," says Baruch Blumberg, a Nobel prizewinner who was appointed director of the NASA Astrobiology Institute in 1999 and is special adviser to the administrator of NASA for biology. "It offers an opportunity to explore areas we never even thought were possible."
Making spare parts for the human body could become a reality. "There is no question whatsoever that in this century we will be growing organs to order from a person's own tissue, and we are providing the key pieces to the puzzle of tissue engineering now," says astronaut and physician Wolf. "To layer the cells correctly and allow them to grow and differentiate and then relayer, we need perfect control over the fluid-dynamic environment. Space and rotation allow us to operate in a heretofore unachievable range of these parameters."
Goodwin predicts this work could also lead to more effective techniques for gene therapy, both in space and on the ground. "If we can learn to manipulate the genome in the microgravity environment in the way we want, then this could become a new process for delivering or inserting a desired gene into a targeted genome. That could result in a global technology applicable to developing treatments for any number of diseases."
Ultimately, the research may explain why some genes are so active in space and even, perhaps, something about our own origins. Goodwin has a bold hypothesis: The signals first seen by cells in the embryo, in a semiweightless environment, may be recalled when the cells are sent into microgravity.
"Our anecdotal evidence is that these cells do seem to be responding in the way that they would if they were embryonic cells," he explains. "They tend to grow well. They don't seem to undergo as much programmed cell death as you see in a mature cell. And they tend to go through proliferative stages, just like embryonic cells. It may be that signals seen by the cells during their embryonic state are being recalled when they're sent into microgravity, because that semiweightless environment is a force of some kind."
An even more provocative possibility is that some of the unusual responses of genes in space could be recalled responses from environments that existed millions of years ago, environments that no longer exist and that perhaps were not even on this planet. In other words, the clues to our very origins lie locked inside our genes, waiting to be discovered.
"There are hypotheses that life may have come to Earth from elsewhere; that is, it might have had the experience of being in zero gravity before," says Blumberg. "If you study contemporary gene expression, some of what you see may be a gene retained from those happy days in space."
"People are not realizing what space science has to offer yet, but it's a whole new landscape for science," says Goodwin. "The intriguing thing is that our investigations into cells and genes in space may reveal something even more astonishing, that while we are viewing space as the new frontier for biology, it just may also be the frontier of our distant past."
For an overview of NASA's microgravity-related projects, visit microgravity.msfc.nasa.gov. For more details on the Bioreactor, see science.msfc.nasa.gov/ newhome/br/bioreactor.htm.
Tulane University devotes a Web page to their astrobiology projects and related links: www.som.tulane.edu/ astrobiology/default.htm.
