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     Outside the OR, Csete puts her knowledge of different gases and their potent effects on living cells to good use when she assumes her other role—that of a pioneering stem cell researcher. She has turned her expertise into a crusade of sorts, or at the very least, a research passion. As head of the medical school’s new core facility for embryonic stem cell research, she hopes to help turn the promise of stem cell therapies into reality.
     Last fall, she received the go-ahead to set up the facility, which will supply human embryonic stem cells grown under tightly controlled conditions to researchers at Emory and those with the joint Georgia Tech/Emory Center for the Engineering of Living Tissues (GTEC). Stem cells will not be derived at the facility. But existing stem cell lines, already approved for use in federally supported research, will be maintained there. The core facility will also serve as a resource center, providing technical assistance and education to investigators conducting various stem cell research projects. Emory and Georgia Tech are in the process of purchasing existing cell lines developed at other institutions.
     In collaboration with research partners at the University of Georgia, Csete has also applied for an NIH grant to fund research on maintaining stem cell lines. UGA scientists led by Steve Stice have been studying these cells since the NIH first approved research with existing stem cell lines several years ago, and Csete’s part of the project would focus on creating the ideal gaseous environment to reduce oxidative stress and lower rates of cell mutation.
     For Csete, the research is more than a scientific endeavor.
     “As physicians, we owe it to our patients to do this kind of research,” she says. “Most people think of stem cells as a resource for replacing cells that have been damaged, which is the Christopher Reeve/Michael J. Fox approach. But in addition to that, working with stem cells gives us our first view into the earliest events of human development. We know tons more about mouse development than we do about human development. Stem cells also may serve as remarkable tools for screening drug candidates, modeling diseases, promoting certain kinds of cell differentiation, and researching protection against cell death.”
     Embryonic stem cells, the primitive, undifferentiated cells that have the potential to develop into any kind of cell in the body, have been the subject of heated public debate because they must be extracted from human embryos. (See On Point) But glaring headlines aside, the biggest obstacle blocking new cell-based therapies may be the difficulty of successfully growing these cells in a laboratory.
     Developing an ideal environment is key to keeping the cells viable, Csete believes, but few researchers have been interested in this area. Though she has rattled the nerves of more than a few other scientists with her quest, she hopes her unique focus will have an important impact on this burgeoning field of study.
     In 1996, Csete interrupted a decades-long career as a liver transplant anesthesiologist at the University of California-Los Angeles, entering Caltech as “the world’s oldest graduate student” to pursue an interest in muscle stem cells, known as “satellite” cells. She began with the hypothesis that gases have a significant impact on living cells, no matter where they are. During her four-year stint in the laboratory, she was shocked to discover that research biologists were not very conscious of the effects of gases on cell culture, using everyday room air for their experiments. She knew that each organ has its own ideal oxygen level, but when she asked her laboratory colleagues if they knew the body’s normal oxygen concentration for their organ of interest, she found not a single faculty member with an answer.
     “This told me right away that my hypothesis was going to be a hard sell,” recalls Csete. “When I first started asking the question, everyone was very upset because they were concerned that their tissue-culture work going back for decades might not be quite right.”
     At the time, only two areas of biology took gases into consideration. Vascular biologists knew that when an organ’s oxygen supply is lowered to dangerous levels, vascular cells take up the slack by proliferating and making new blood vessels to deliver more oxygen. Models of vascular biology have for decades included very low oxygen conditions. Cancer biologists know that as tumors outgrow their blood supply, the center of the tumor becomes very low in oxygen, a condition called hypoxia. The cells that survive hypoxia become the most dangerously resistant to chemotherapy and radiation. In some ways, says Csete, these hypoxia-regulated cells turn into cancer stem cells.
     Armed with a PhD from Caltech, Csete then moved to the University of Michigan, where she continued her work with oxygen and cell culture, using a specially designed work area where gases could be controlled during long-term stem cell growth. She began to ask the simple question of what happens to stem cells when you change
the gases around them to reflect not room air, but the exact gas concentration cells actually encounter in the body. She was not surprised to discover that a particular balance of gases made a tremendous difference to growing cells.
     “We found that cells are happier and healthier and that they live longer in oxygen concentrations that reflect the level in the body of 2% to 5%,” Csete explains. “Maintaining concentrations in this range is tricky, especially over long periods of time, but it’s a very powerful tool. I would say with conviction that based on the kinds of changes we see with different oxygen levels, you have to be very rigorous and never let in room air.”
     Stem cells are very similar to cancer cells in their tendency to proliferate and migrate, Csete explains, which is why it is critical to eliminate mutations while growing the cells. Embryonic stem cells are particularly vulnerable to chromosomal breaks and severe mutations. Although cells with damaged chromosomes are naturally programmed to die, those few that manage to survive would have an enormous potential to reproduce in a tumor-like fashion.
     “That’s why, at this point, you would not consider putting a completely undifferentiated stem cell into a human until there are better ways of predicting how to control their properties. And any changes in the growing environment of embryonic stem cells will lead them to differentiate, so conditions must be maintained very carefully.”
     The stem cell core facility consists of just two tiny laboratories on campus. But the small space embodies Csete’s zealous belief in keeping the cells protected from room air. The cells here will be stored and cultivated in protective mini-incubators and exposed to very low levels of oxygen ranging from 1% to 8%. Csete’s prior research showed that under oxygen conditions that mimic conditions inside the body, stem cells can be protected from undergoing chromosomal damage.
     “There is nothing more fundamental in biology than oxygen,” Csete emphasizes. “As anesthesiologists, we’re conscious right up front of what gases do. We monitor them in the operating room. But for most biologists, the gas context is just not there. It’s not a major part of what they are taught. Students who jump right into molecular biology to study how genes interact inside cells don’t get the bigger picture that using room air for experiments is aphysiologic.” Csete says. “There is a huge bank of historical literature from scientists who have grown cells in room air because of convenience, and that practice is hard to change because there are thousands of papers already written based on that method. The vast majority of laboratories still have incubators that rely on room air, where the oxygen concentration is 20%.”
     Emory and Georgia Tech researchers are uniquely qualified to study the interaction of embryonic stem cells with the environment, says Csete.
     “Our research—which includes novel approaches to studying mechanical influences on the cells and also the effects of signaling from the extracellular matrix and the effect of the gases—is very likely to lead to a better understanding of the therapeutic roles of human embryonic stem cells for a wide variety of diseases.”

Holly Korschun is the director of science communications at the
Woodruff Health Sciences Center.

Stem Cells 101: A Primer
by Marie Csete
Rarely have science, politics, and medicine collided in the public imagination as they have over the stem cell debate. Yet despite all the air and press time, the average person still has only a vague idea about what these cells are and where they come from.
     First, the basic term “stem cell” is used to describe a number of types of undifferentiated cells that have the potential to become different kinds of specialized cells. Stem cells function as a kind of repair system for the body. By themselves, they lack the potential to function physiologically, that is, to carry out specific bodily tasks. For example, stem cells cannot act as neurons because they do not express the signaling chemicals that neurons use to communicate. They cannot act as muscle cells because they do not express the contractile proteins necessary for muscle activity.
     They have the ability, however, to make cells (called daughter cells) of more than one type. For example, certain blood stem cells can generate platelets, white cells, and red blood cells. Scientists call this ability to generate diverse cell types multipotentiality. Potency in stem-cell language refers to a cell’s potential to generate different specialized cell types. Multipotent stem cells can generate just a few different kinds of cells. Pluripotent stem cells can generate many different kinds of specialized cells. Totipotent stem cells have the potential to generate any cell type in the body, including germline cells (sperm and egg). Differentiation is the process by which stem cells mature and change into physiologically functional, specialized cells.
     While multipotent and pluripotent stem cells exist in the human body in varying degrees throughout a person’s lifetime, totipotent cells are found only in embryos in the earliest stages of development. The totipotency of embryonic stem cells is the reason for scientific excitement about these cells, but the totipotency also means that the control of differentiation toward just a single type of specialized cell is technically difficult, because all the other options for differentiation have to be negatively controlled.
     Stem cells also have the ability to self-renew, a property that is unique to them. When stem cells divide, the new cells can be either new stem cells or more specialized types of functional cells. In some cases, stem cells also may undergo asymmetric division, in which one cell retains its stem cell-ness and the other goes on to develop into a specialized cell.
     People are often confused by references in the media to “adult” and “embryonic” stem cells.
     Adult stem cells are derived from tissue sources that have already differentiated. “Adult” here does not mean from a person who is past puberty. Adult stem cells can be derived from fetal sources, young people,
or adults.
     Embryonic stem cells are derived from a single cell removed from a fertilized egg that has been maintained in a lab and allowed to divide to a particular embryonic stage called the blastocyst. Embryonic stem cells from the lines approved for research using federal funds come originally from blastocysts left over from in vitro fertilization procedures designed to help infertile couples have children.
     Only embryonic stem cells are considered totipotent, whereas adult stem cells are more restricted in the kinds of specialized cells they can generate. In general, stem cells are harder to find and grow when derived from older organs and tissues, and they generally lose some of their ability to be stem cells with long times in the body. The aging of stem cells is the focus of work in our lab.
     Researchers still have much to learn about stem cells and how they function, including how stem cells might be manipulated to treat many different illnesses and medical conditions. But many scientists believe these cells hold the potential for great medical and scientific advances.
     If you want to know more about stem cells, the National Institutes of Health maintains a website devoted to stem cell research. It can be found on the web at this address:

Marie Csete is assistant professor of cell biology and the John E. Steinhaus Professor of Anesthesiology in the School of Medicine.



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