Microgravity and Gene Expression: Early Results Point to Relationship

It's a simple but haunting question: Can microgravity influence gene expression? Yes, according to a recent study of human cells conducted aboard NASA's space shuttle. In fact, the results are so promising that the investigators believe their continuing research could lead to better toxicology tests, key elements of tissue engineering, and new treatments for various diseases--while broadening the scope of scientific experimentation. Astronaut Katherine P. Hire works with the bioreader system aboard Columbia on April 28, 1998. "We flew a cell culture incubator containing human kidney cells specifically to look at renal hormone production of vitamin D3 and erythropoietin, and we found that there were dramatic effects, that many, many genes change," says principal investigator Timothy G. Hammond, associate professor of medicine and nephrology at Tulane University Medical Center's Environmental Astrobiology Center and a Veterans Administration (VA) physician. "Our idea was that this [occurred] because the mechanical culture conditions in flight were changed." Unlike conventional cultures on the ground, a microgravity suspension culture permits "the simultaneous achievement of low-fluid shear stress, three-dimensional spatial freedom, and cospatial location of particles with different sedimentation rates," points out coinvestigator David Wolf, NASA astronaut and electrical engineer. "This is the first time that a physical environment has been proven to be responsible for changes in gene expression, and that opens up a whole new area of science, because everything that's been done up to now has been done in a one-gravity environment," contends coinvestigator Thomas J. Goodwin, manager for biotechnology flight definition/cell biology at NASA's Johnson Space Center. "No," counters Wolf, "because we can prove that hitting cells with a hammer affects their genes, and that's a physical environment. Actually, it's the first time we have explored the mechanical environment of those three simultaneous conditions out in the microgravity region. We were the first to explore this region with this many genes, and that's a result of our [space] technology and gene array technology." Although the work to date has been reported only briefly in a letter to Nature Medicine,1 a comprehensive report has been submitted for publication in another journal, according to Hammond. Researchers inoculated the human kidney cells on the ground within three days of placing them into bags and loading them into an incubator, dubbed the Biotechnology Specimen Temperature Controller. Aboard the space shuttle Columbia, the cells became a part of NASA's Neurolab mission in April 1998.2 "The cells then grew for six days to steady state on orbit, with the fixative added by the astronauts on board, Rick Linnehan and Kay Hire," Hammond explains. As a negative control on the ground, the team spun another batch of cells in a centrifuge at three gravities, which doesn't change three dimensionality, bring dissimilar cell types together, or change shear or turbulence. Later, they also inserted the same types of cells into NASA's ground-based, state-of-the-art optimized suspension culture device known as the rotating wall vessel (RWV), which simulates some of the conditions of microgravity. "In each case, we used cells from the same donor, and did standard conventional cultures next to these so we had a uniform control," maintains Hammond. Once Columbia was back on the ground, the cells from the flight were shipped to Hammond's lab at Tulane, where the RNA was prepared, and then on to Incyte Pharmaceuticals in Palo Alto, Calif., for automated gene array analyses. "We looked at 10,000 genes, including every known cloned human gene and many that are just being cloned," Hammond explains. It took about a year for the data to be compiled and written up, "but the very preliminary [findings] indicated that we had some fairly significant results," says Goodwin. In the control experiment on Earth, only five of the 10,000 genes changed during six days at steady state. "So as we predicted, it was a true negative control and confirms our idea that not changing those mechanical culture conditions was relevant as to whether we got changes," Hammond says. In the rotating wall vessel, the researchers found that 914 genes changed; they either were up-regulated or down-regulated. "And in flight, 1,632 genes changed their response--more than twofold--so we found a much bigger spread. A lot more genes are changing in flight, and that is dramatic," Hammond maintains. Many of the changes that occurred made "intuitive sense," he says. "One of the genes that went up the most, for example, is the vitamin D receptor, and the one that went down the most is the gene that controls pediatric tumors, both of which are very well-known, developmental genes in the kidney." Why does microgravity change the expression of genes so dramatically? Says Goodwin, "Our basic hypothesis is this: Gene expressions go through different phases as the embryo develops, and if you look at how the embryo actually develops--in a semiweightless environment, a neutrally buoyant environment--it may be that a lot of the signals seen by the embryo may be recalled by cells when they're sent into microgravity. Now, we have no substantial proof of that, but it would seem from the anecdotal data we have so far that these cells respond in the way that they would respond if they were embryonic cells." "The really interesting part is that the RWV and flight changed different gene pathways, so one is not simply an extension of the other," Hammond explains. On the ground, gravity must always be balanced with other forces, a trade-off that has confounded many scientific efforts. Microgravity, however, offers a unique operating regime. "The bottom line is, we avoided the trade-off," says Hammond. "That ... lets you look at mechanical culture conditions in a hitherto unobtainable area, but more importantly, it lets you get at the mechanism by which the changes are happening, by looking at the groupings of genes." The team is currently negotiating a return trip to space to repeat the experiment and to verify the results. In addition, they hope to begin studying mechanisms by gaining insight into transcriptional events from the time the cells actually enter the microgravity environment through the first 24 hours. "We feel from a molecular genetics standpoint there are significant transcriptional events ... that we don't see once the cells reach the steady state, and we'd like to know exactly what those ... events are so we can map how the genes are promoting the effects that we ultimately see ...," comments Goodwin. Space flights, however, remain expensive, and the shuttle does not allow for extensive experimentation. "The whole point is to not use the shuttle as a factory but to use the information to do better studies on the ground," explains Hammond. "If you can get special features back on the ground, for example, you can use that for your toxicity studies rather than using animals .... We hope this will lead to a better in vitro system for testing toxins on the ground." Developing better toxicology tests is only the beginning. "There's no question that one day humans will grow organs to order, and there's no question in my mind that we will provide key pieces to the puzzle of tissue engineering," asserts Wolf. "Now, how do we layer the cells correctly, and how do we allow them to grow and differentiate and then relayer? We need perfect control over the fluid dynamic environment to do that, and it's clear to us that microgravity offers the ultimate ability to control the mechanics of the environment to organize and grow tissue. You just can't get these structures to form in Earth-based systems." Wolf has seen the possibilities up close. In a predecessor experiment to the one on Columbia's Neurolab, he took an incubator on the Russian space station Mir and grew nerve tissue with rat renal tubular cells from the kidney. "It grew in delicate, frondlike structures that were vastly different from conventional culture on the ground and different from the rotating wall vessel," Wolf says, adding that there was "a spectacularly different organization" of the cells. In the case of kidney cells, "we got a very fluffy, large organizing tissue, and in fact, the whole bag turned into one piece of tissue; whereas on Earth, what you see are individual cells or small clumps, and you don't see them organizing into one network." Wolf is quick to point out that he doesn't believe there's anything "magical" about microgravity. "You have to look at each culture system you're using as a ground control and look at it in terms of these mechanical properties you achieve in gravity or out of gravity. I am a strong proponent that we need to interpret all of these experiments not in terms of a magic word 'gravity effect,' but in terms of how gravity couples to the mechanics of the system." Causal relationships between the mechanics and the ultimate gene display and morphology can then be determined. "And that is fundamental knowledge for tissue engineering. I compare it to semiconductor fabrication 30 years ago, learning to lay down the three-dimensional semiconductors we use in integrated circuits.... We're laying down cells ... trying to allow their genetic predisposition to be expressed as a tissue." John R. Feussner John R. Feussner, chief of research and development for the VA in Washington, D.C., who has reviewed the work, was pleasantly surprised with the results. "Quite frankly, this whole line of research beginning with Mir and through Neurolab has been remarkable to me, and I, for one, was not anticipating the research results would be so rich .... I don't think it's serendipity; rather, it's a credit to the creativity and innovation of these investigators." The VA partially funded the study, largely because of its interest in research on chronic disease. Although this research probably won't be applicable for some years, "it's the kind of work that's necessary if we're ever going to solve some of these problems that cause failure of critical organs ..., and the time to start that research is now," says Feussner. "These experiments may well be viewed 10 to 20 years from now as the initial steps taken in that classical scientific process of discovery, when we figure out what makes a cell organize itself differently and what the genetic components are that have to be turned on or turned off." Wolf says the desire is to be able to grow tissue on the ground, but "by understanding the principles of what's important, we can cheat and do it in space first .... Hopefully we can mimic that and do it on the ground, but it could also form the basis of a future industry in space." In a moment of "wild speculation" about potential applications, Goodwin ventures even further: "If we can actually manipulate the genome in the microgravity environment in the way in which we want to manipulate it specifically, then we can speed or slow, perhaps, genetic incorporation of different kinds of genes to effect different kinds of results. There may be a vast area where we can use this physical environment to expedite and control gene expression at the same time we're effecting change. That means that ultimately this technology could be applicable to developing treatments for any number of diseases. It may well be a global technology, like PCR--that wide ranging. Still, this is speculation." In any case, there's "a new sheriff in town," Goodwin declares. "There isn't a scientist in any standard laboratory in the United States who considers gravity as a variable in his or her research, and they don't because gravity is not something they can modulate. Well, now we have shown that gravity can be modulated." Moreover, when the space station is in operation, Goodwin continues, infinitely exquisite amounts of gravity can be induced back in microgravity: "We can go from a half a gravity to three-quarters or down to 0.25 gravity simply by spinning things in a centrifuge in a microgravity environment. So we have a process now by which we've proven this environment [has a definitive effect], and we can modulate that environment to elicit the kinds of responses we may want to see." The space station will provide the area necessary for the laboratory to develop the principles, Wolf concurs. "You need true laboratory facilities to operate a lab as you would on Earth so you can make mistakes, learn from them, and then redo experiments, and the space station will provide that." At this stage, however, it has yet to be truly determined whether even space offers ultimate advantages. "We have very tantalizing results from space that show we can build these structures," says Wolf. "That we can grow these structures from these cells in vitro ... means we really should be able to do that on the ground." Hammond concludes: "It just might be that we're doing something worthwhile that actually will help people here on Earth. Now isn't that a radical idea?" A.J.S. Rayl (ajsrayl@loop.com) is a freelance writer in Malibu, Calif. T.G. Hammond et al., "Gene expression in space," Nature Medicine, 5:359, April 1999. S. Bunk, "Global cooperation enhances space flight research," The Scientist, 12[12]:10, June 8, 1998.

Microgravity and Gene Expression: Early Results Point to Relationship

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