Tissue engineering is an emerging interdisciplinary field that applies the principles of biology and engineering to the development of viable substitutes that restore, maintain, or improve the function of human tissues or organs. This form of therapy differs from standard therapies in that the engineered tissue becomes integrated within the patient, affording a potentially permanent and specific cure of the disease state. Three general approaches have been adopted for the creation of new tissue: 1. Design and grow human tissues outside the body for later implantation to repair or replace diseased tissues. The most common example of this form of therapy is the skin graft, which is used for treatment of burns. Skin graft replacements have been grown and used clinically for more than 10 years. 2. Implantation of cell-containing or cellfree devices that induce the regeneration of functional human tissues. This approach relies on the purification and large-scale production of appropriate "signal" molecules, like growth factors, to assist in biomaterialguided tissue regeneration. In addition, novel polymers are being created and assembled into three-dimensional configurations, to which cells attach and grow to reconstitute tissues. An example is the biomaterial matrix used to promote bone re-growth for periodontal disease. 3. The development of external or internal devices containing human tissues designed to replace the function of diseased internal tissues. This approach involves isolating cells from the body, using such techniques as stem cell therapy, placing them on or within structural matrices, and implanting the new system inside the body or using the system outside the body. Examples of this approach include repair of bone, muscle, tendon, and cartilage, as well as cell-lined vascular grafts and artificial liver that are being developed in Pittsburgh. Broadly defined, tissue engineering is the development and manipulation of laboratory-grown molecules, cells, tissues, or organs to replace or support the function of defective or injured body parts. Although cells have been cultured, or grown, outside the body for many years, the possibility of growing complex, three-dimensional tissues - literally replicating the design and function of human tissue - is a recent development. The intricacies of this process require input from many types of scientists, including the problem solving expertise of engineers, hence the name tissue engineering. Tissue engineering crosses numerous medical and technical specialties. Cell biologists, molecular biologists, biomaterial engineers, computer-assisted designers, microscopic imaging specialists, robotics engineers, and developers of equipment such as bioreactors, where tissues are grown and nurtured, are all involved in the process of tissue engineering. Tissue engineers in the United States and abroad have set out to grow virtually every type of human tissue - liver, bone, muscle, cartilage, blood vessels, heart muscles, nerves, pancreatic islets, and more. Commercially produced skin is already available for use in treating patients with diabetic ulcers and burns. Many current medical therapies may be improved upon by tissue engineering with significant financial savings. Take organ transplantation, for example. In standard organ transplantation, a mismatch of tissue types necessitates lifelong immunosuppression, with its attendant problems of graft rejection, drug therapy costs, and the potential for the development of certain types of cancer. Furthermore, there is always the potential for rejection of the tissue, and the surgery, itself, always carries some risk. As the field of tissue engineering progresses, it will inevitably provide many improvements: The costs of tissue harvest and postoperative patient costs will be significantly reduced. By actually designing replacements to mimic the native tissue being reconstructed, the adequacy of tissue function will be optimized, leading to improved patient care at less expense. A Compelling Need By Ricki Lewis Field Author: Ricki Lewis Sidebars: A Tissue Survey Over the past decade, tissue engineering has evolved from a hodgepodge of different disciplines to a biotechnology field in its own right. A marriage of chemical engineering and cell biology, with input from genetics and surgery, tissue engineering combines living cells, biochemicals, and synthetic materials into implants that can function in the human body. Some 30 companies and dozens of academic laboratories are pursuing tissue engineering, with the ultimate goal of fashioning stand-ins for such varied tissues as pancreatic islets, liver, skin, cartilage, bone, nervous tissue, bone marrow, and blood vessels. And although experts agree that it will still be a few years until the family physician offers up replacement skin, engineered tissues are already impacting basic cell culture research, taking it one step closer to mimicking true in vivo conditions. Today's tissue engineers are an eclectic group. The field "includes chemical engineers, chemists, cell biologists, and surgeons. Many times you find teams, or people who can work in several areas at once," observes Jeffrey Hubbell, a professor of chemical engineering at the California Institute of Technology. Hubbell investigates synthetic polymers that can serve as scaffolds in engineered tissues. "People have had the idea for years to generate new tissue, but they have not had the techniques, nor new synthetic materials, to make that a reality," notes Charles A. Vacanti, chairman of the department of anesthesiology at the University of Massachusetts Medical Center in Worcester. He is an editor of Tissue Engineering, a new journal published by New York-based Mary Ann Liebert Inc. He is also a cofounder of a new professional organization, the Tissue Engineering Society, with his brother Joseph Vacanti, a transplant surgeon at Children's Hospital in Boston, and Robert Langer, a professor of chemical and biomedical engineering at the Massachusetts Institute of Technology. Charles Vacanti says he was stimulated to enter the field by an idea to combine polymers and cells that his brother and Langer were tossing around eight years ago. His experience as an anesthesiologist helped him recognize the need for engineered tissue. "I would peek over the shoulders of orthopedic surgeons and watch them use bone grafts and bone powders," he recalls. "I began thinking about my brother and Robert Langer's idea to create new tissue." Charles Vacanti began working in Langer's lab at night, starting with cartilage. "Since then, we've worked on about 10 different tissues," says Langer. Motivating researchers in the field is the great demand for human spare parts. The cost stemming from treatment and lost work hours for tissue and organ disease and injury in the United States per year exceeds $400 billion, according to Langer and Charles Vacanti. And despite the speed with which baseball hero Mickey Mantle and rock musician David Crosby obtained liver transplants, fewer than 3,000 livers become available in the U.S. each year - as 30,000 people die from liver failure. The numbers are similar for other organs and tissues. Even the few existing organ replacements or alternatives, such as skin grafts, ventricular-assist devices, and kidney dialysis, are hardly ideal. While clinical trials for some engineered tissues progress, the cell-synthetic combinations are also finding uses in basic research. "In my lab, our long-term goal is to develop a blood vessel substitute, but our initial goal is to develop a better model for use in cell-culture labs," says Robert Nerem, a professor of mechanical engineering at the Georgia Institute of Technology. Such hot cell biology areas as signal transduction, cell adhesion, and carcinogenesis will benefit from engineered tissues, which can allow researchers to isolate steps in these complex processes. For example, several researchers are manipulating a three-amino acid sequence (arginineglycine-aspartic acid), called the RGD sequence. This tripeptide is part of a common cell-surface receptor protein which, like Velcro, enables cells to stick to each other and to an extracellular matrix (substances cells secrete that surround them). By incorporating the RGD sequence into synthetic polymers, chemical engineers can add a Velcro-like touch that attracts certain cells. "We sometimes add the RGD sequence to our synthetic biodegradable polymers," notes Langer. "People are finding that key amino acid sequences can be used to regulate cell growth. A lot of information is apparently packed into a few amino acids." Like many areas of biomedical engineering, tissue engineering is a field that existed before it had a formal title. Many review articles date its formal debut to a series of National Science Foundation meetings. First was a panel meeting early in 1987 when the name was brought up. Then, in October, a forum on emerging technologies identified tissue engineering as an area for rapid industrial development. At an NSF workshop on tissue engineering held at Lake Tahoe on Feb. 26-29, 1988, a formal definition was drafted: "Tissue engineering is the application of principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve tissue functions." But long before tissue engineering had a name, researchers in more traditional fields were experimenting with teaming cells and synthetic materials. Carl Wolf, director of the Blood Bank Transfusion Service at New York Hospital Cornell Medical Center, a chemical engineer-turnedphysician, was one of them. Before and during medical school, in the late 1960s and early 1970s, he worked at the DuPont Experimental Station in Wilmington, Del. At first, he was combining hollow synthetic fibers conduits for nutrients and wastes - with kidney cells to mimic renal function. Then, when blood clotting became a problem, he had the idea to add liver cells, which secrete the anticoagulant heparin. Realizing that the hollow fibers with liver cells could emulate liver function, he turned his interest toward that organ. Later he found a hepatitis research group at the New York Blood Center that was growing liver cells, and he went to work on a bioengineered liver. "At first I tried growing cells in fibers, lining them, with only moderate success. It occurred to me that maybe cells could function just as well growing on the outside of the fibers - which is the geometry that is currently being tried clinically," says Wolf. Great ideas sometimes spark in several minds at once. At the same time that Wolf was nurturing his liver hollow-fiber bioreactor, Richard Knazek at the National Institute (now Institutes) of Health was doing the same thing, and patented the invention (R.A. Knazek, Science, 178:65, 1972). And at the University of Massachusetts Medical School, William L. Chick was combining insulinsecreting pancreatic islet cells with hollow fibers to treat diabetes in dogs (W.L. Chick, Diabetes, 20:327, 1971; W.L. Chick, Science, 187:847, 1975). Then came AIDS, and Wolf had to put his research interests aside as he dealt with the mounting crisis of HIV-contaminated blood. "That put an end to my research in tissue engineering," he says. "I advanced the field as far as I could. I showed how to grow cells on hollow fibers, and how to hook them into animals. But the field needed a major developmental effort to take it from research to engineering." That push came in the mid-1980s, when tissue engineering began to find its niche in the biotech industry. Organogenesis Inc. of Canton, Mass., was formed in 1985, and Advanced Tissue Sciences Inc. in La Jolla, Calif., in 1986. Both companies are racing to market their skin substitutes. Providence, R.I.-based CytoTherapeutics came on the scene in 1988, and focuses on central nervous system implants (see accompanying story). Several other companies are also pursuing tissue engineering. Building tissue entails several steps. The first is biological - identifying cell types that constitute a tissue. "The biggest challenge is to get the right structure for a tissue with many cell types," Hubbell points out. "For example, cartilage is easy, because it is not vascularized, and consists of chondrocytes [cartilage cells] in a matrix that's not highly organized. Skin also has a relatively low structural level. Larger, vascularized constructs, such as pancreas, liver, and kidney, are more difficult to model." Researchers must also select synthetic materials, usually polymers, that are biodegradable and compatible with cells. In addition, cells are often stripped of surface molecules that could stimulate an immune response. Synthetics have molecules added that can foster cell adhesion. A step less studied in tissue engineering is to understand the physical constraints on tissue formation and maintenance. How will pressure from surrounding structures affect implanted tissue? How can a tissue be engineered so that each cell is only a short distance from a blood supply, as occurs in vivo? How do shear and tension affect cell specialization? These considerations are where engineering comes in. Looking at tissue structure and function from an engineering perspective enabled J.H. David Wu, an associate professor of chemical engineering, microbiology, and immunology at the University of Rochester, to develop a long-term bone marrow culture that may make bone marrow transplants easier, cheaper, and more successful. The procedures are performed to treat inherited or acquired blood disorders or to increase tolerance to chemotherapy and radiation therapy. Wu noticed that the conventional way to culture bone marrow cells - in petri dishes or culture flasks - was nothing like the real thing. So he took cues from the anatomy of bone marrow. "In the body, bone marrow is a spongy filler of bone," Wu explains. "When blood flows through bone marrow, it takes with it mature blood cells, which egress into blood vessels from bone marrow. In the body, bone marrow makes about 10 different types of blood cells. But in a petri dish, it makes only two." Wu developed a bioreactor that nurtures bone marrow cells in a sponge-like material consisting of porous bovine collagen microspheres. "With this method, you start to see all the cell types. By taking an engineering approach, we are moving into a completely different way to culture bone marrow," he says. An enthusiastic response to the new journal and society are evidence of the field's growth, according to Vacanti. Using lists from existing societies to which people doing this research might belong, as well as by searching citations, Vacanti sent postcards to 1,000 people, inquiring about interest in forming a society. Within weeks, he had 500 responses. With his brother and Robert Langer, he began planning the Tissue Engineering Society. "I found very quickly that there was a big need for the journal and, talking to people at national meetings, also learned there was a big need for a society," he says. Even though engineered tissue as a treatment isn't yet a medical reality, there are already benefits for life scientists. Concludes Robert Nerem: "Tissue engineering won't enter the clinic for a decade, except perhaps for skin. But in the meantime, there will be significant payoff in terms of the impact of these constructs in the ability to do basic research. What we are seeing is the very beginning of an industry." Ricki Lewis is a biology textbook author and geneticist based in Scotia, N.Y. (The Scientist, Vol:9, #15, pg.12 , July 24, 1995) (Copyright © The Scientist, Inc.) [email protected] RESEARCH Stem Cells Tapped to Replenish Organs Embryonic or adult? The superior source depends on the tissue By Douglas Steinberg Credit: Eric Laywell Editors Note: This is the second of two articles on issues raised by recent stem cell discoveries. The first article appeared in the November 13 issue "All politics is local" was a famous maxim of Thomas "Tip" O'Neill, the late speaker of the House of Representatives, and the same can be said of medically useful stem cells. Progenitor cells may prove to be more or less pluripotent in the lab, but if they don't succeed on a local level in the body, they won't cure anything. They must be capable of being coaxed into differentiating reliably into the cell types that populate particular organs. How much can embryonic stem cells (ESCs) and adult stem cells (ASCs) replenish tissues of the brain, pancreas, liver, heart, and blood? So far, researchers have manipulated ESCs to generate a broad span of cell types. ASCs have yielded a narrower range, partly because several subtypes haven't been isolated yet. The phenomenon of transdifferentiation, however, promises to extend the capabilities of ASCs. And as studies proliferate in the wake of discoveries and the issuance of new guidelines by the National Institutes of Health, the relative advantages and disadvantages of ESCs and ASCs could change considerably An astrocyte monolayer that can be coaxed into becoming within the next few years. multipotent neural stemlike cells Brain Goal: To replace neurons that have died as a result of degenerative diseases or stroke. Ronald D.G. McKay and his Laboratory of Molecular Biology at the National Institute of Neurological Disorders and Stroke can efficiently generate dopaminergic and serotonergic functional neurons in vitro from mouse ESCs.2 They can get ASCs, in the form of mesencephalic precursor cells, to induce functional recovery when transplanted into parkinsonian rats. 3 But according to McKay, these ASCs stop generating dopaminergic neurons in culture after a week or so. The yield improves if the cells are grown under low-oxygen conditions, which are characteristic of the fetal environment.4 Still, McKay notes that his lab's experience thus far with several types of ASCs is that "they don't turn into dopaminergic neurons with any kind of efficiency." Referring to a 1999 paper from the Karolinska Institute that reported such a result,5 he wonders whether the final yield is "really a dopaminergic cell or not." One problem besetting such research is the uncertain identity of ASCs in the mammalian brain. Last year, a Karolinska team led by Jonas Frisén announced that the ependymal cells lining the brain's ventricles were neuronal ASCs.6 Five months later, a Rockefeller University group headed by Arturo Alvarez-Buylla countered that subventricular zone (SVZ) astrocytes were the true neuronal ASCs. This group also rejected the ependymal-cell hypothesis after finding that those cells neither formed neurospheres, nor accumulated nucleoside labels, as they would if they divided.7 The New York Times ran a story on the ensuing brouhaha.8 Credit: Eric Laywell Alvarez-Buylla, who just moved to the neurosurgery department of the University of California, San Francisco, says that the conflict may arise, in part, because SVZ astrocytes "interact very, very closely with the ependymal cells." But he maintains that ependymal cells only serve to create a niche where neurogenesis can occur. His lab is currently examining two signaling systems that seem to prompt SVZ astrocytes into becoming neurogenic.9 Last June, Frisén bolstered his theory with a paper showing that neural stem cells had broad differentiation potential.10 The authors couldn't verify that most of their experiments actually involved ependymal cells. But when ependymal-cell-derived neurospheres were injected into the amniotic cavities A clone of newly generated neurons derived of chick embryos, the cells showed broad differentiation potential (the data, at footnote 16, from a monolayer of astrocytic stemlike weren't published). Frisén now says he has additional, cells unpublished lines of evidence indicating that ependymal cells are neural stem cells. His theory may need that support. Derek van der Kooy and his colleagues at the University of Toronto weren't able to get ependymal cells to make neurons in vitro.11 A similar negative finding appears in an upcoming paper describing a study led by Eric D. Laywell and Dennis A. Steindler, professors of anatomy and neurobiology at the University of Tennessee in Memphis.12 They and their colleagues, on the other hand, confirmed Alvarez-Buylla's hypothesis by observing that SVZ astrocytes could give rise to neurons, as identified by the expression of ß-III tubulin and other markers. (Functional studies of the neurons are now under way.) In a significant extension of that hypothesis, they found that astrocytes from cerebral cortex, cerebellum, and spinal cord could also turn into neurons--but only if the astrocytes were derived in the first two postnatal weeks. "This correlates with what we believe to be the maturation of the astrocyte in the nervous system," notes Steindler. "The end of this critical period in astrocyte multipotency coincides with the end of a period in which the brain's regenerative responses are far more successful than those in the more mature brain." Pancreas Credit: Russ Lante, University of Florida, Health Goal: To replace insulin-producing islet ß cells destroyed in Sciences Center some types of diabetes. Stem cell research involving the pancreas seemed to score two home runs this year. In February, Bernat Soria and colleagues at the Universidad Miguel Hernandez in San Juan, Spain, reported that they had obtained insulin-secreting cells from mouse ESCs by using antibiotic selection under the control of the insulin gene's regulatory regions.13 Soria says he is now trying to replicate his results using human ESCs. (A poster at a recent diabetes meeting, meanwhile, is said to have announced that human ESCs differentiate spontaneously into insulin-positive cells.) A month after the Soria paper came out, a team of researchers led by Ammon B. Peck, a professor of pathology, immunology, and laboratory medicine at the University of Florida College of Medicine in Gainesville, reported a second major advance. They claimed to have reversed diabetes in non-obese diabetic (NOD) mice by transplanting islets generated in vitro from pancreatic ASCs, which had not been previously isolated.14 NOD mice are the best current model for autoimmune diabetes. Nora D. Sarvetnick, a professor of immunology at Scripps Ammon Peck Research Institute, is puzzled by Peck's results. "Unless you immunosuppress the mouse"--which wasn't done--"the mouse is just going to reject the ß cells," she contends. Peck responds that cells grown in culture, such as his ASC-derived islets, sometimes exhibit lower antigenicity for unknown reasons. Credit: J. Berndt Susan Bonner-Weir, an associate professor of medicine at Harvard Medical School, objects that the amount of insulin in Peck's ASC-derived islet cells was "orders of magnitude" too low. "What they were putting in [the NOD mice] would have been a very minuscule amount," she says, though she concedes that more insulin might have been made if the islet cells differentiated further inside the mice. BonnerWeir's own work involves expanding human pancreatic duct cells in vitro, then turning them into insulin-producing islet cells.15 She calls the duct cells, which are differentiated, "functional stem cells" because they undergo scores of doublings in culture Susan Bonner-Weir and help to regrow pancreas after a portion is removed. Liver Goal: To develop a plentiful source of hepatocytes for regenerating damaged livers and treating some metabolic diseases. Another functional stem cell is the hepatocyte. "For liver repopulation purposes and transplantation, the best cell type is the differentiated hepatocyte," says Markus Grompe, a professor of molecular and medical genetics and pediatrics at Oregon Health Sciences University. He adds that in transplants, hepatocytes are "far superior" to liver stem cells, whose existence has been established only in the past 12 months or so. The major source of hepatocytes for therapeutic purposes, however, is human cadavers. More accessible and plentiful are the liver stem cells residing in the bone marrow, discovered by Neil D. Theise, an associate professor of pathology at New York University School of Medicine, and colleagues. Their proof: The Y chromosome pops up in some hepatocytes after male marrow transplants into females. Are these new liver cells functional? In a small-scale study of human transplants,16 "We show such extensive engraftment that it's hard to avoid the conclusion that this is a part of physiological regeneration," asserts Theise. The next test is to use bone-marrow transplants to correct defective liver function in animal models of some human metabolic diseases. Grompe and a team of researchers published a paper this month reporting such a finding in a mouse model of tyrosinemia.17 The roles played by ASCs in the liver are still far from clear. Intrahepatic oval cells have recently-and grudgingly--won full acceptance as stem cells, particularly after injury. (Theise proposes that oval cells ultimately derive from the bone marrow.) Apparently no one has yet generated liver cells from ESCs. The growth factors "are just absolutely not known," notes Grompe. Heart Goal: To replace cardiomyocytes that have died during heart attacks. Several years ago, the lab of Loren J. Field, a professor of medicine and pediatrics at Indiana University School of Medicine in Indianapolis, derived relatively pure cardiomyocyte cultures from transfected mouse ESCs.18 The cardiomyocytes weren't identical to their adult counterparts. But according to Field, experimental data suggest that under appropriate humoral and neuronal stimulation, a cardiomyocyte derived from ESCs "will adapt the characteristics typical for the adult cell." Transdifferentiation Researchers will have to understand transdifferentiation better before they can The number of heart muscle cells in a mouse is several orders of magnitude lower than the number in a human. Now that his lab has refined its methods, Field is optimistic that "with bio-processing and growth factors, we can produce sufficient cells for therapeutic applications." To address the low efficiency at which the cardiomyocytes seed into recipient hearts, he is testing such strategies as blocking apoptosis, making the cells more resistant to ischemia, and boosting their capacity to divide. Geron Corp., based in Menlo Park, Calif., and a few academic labs have already shown that cultured human ESCs can give rise to cardiomyocytes. Meanwhile, the presence of ASCs in the heart itself still hasn't been proven. "If they exist, they aren't doing their job," Field says, noting the heart's limited capacity to heal after injury. Other researchers have reported finding ASCs for cardiomyocytes in other parts of the body such as the bone marrow, but no such claim has yet won wide acceptance. deploy adult stem cells (ASCs) as broadly and Blood Richard C. Mulligan, a professor of genetics at effectively as possible. Transdifferentiation is the phenomenon whereby a muscle ASC, say, can give rise to a blood cell. Margaret A. Goodell, who studies stem cells at Baylor College of Medicine's Center for Cell & Gene Therapy, foresees that once biologists begin to "rationalize" the recent spate of observations of this phenomenon, "it won't turn out to be just this wild free-for-all where anything can differentiate into anything." Rules discovered over the last 20 years, she adds, "must have some meaning because otherwise you wouldn't get the development of a very highly organized animal." Harvard Medical School, has proposed Goal: To develop a limitless source of blood cells for transfusions. Over the past 30 years, a small army of researchers has investigated the culture conditions under which hematopoietic ASCs preferentially give rise to myeloid or lymphoid lineages. (Relatively pure cultures of red blood cells have been the most elusive to produce.) Gordon Keller, a professor at Mount Sinai School of Medicine's Institute for Gene Therapy and Molecular Medicine, has succeeded at differentiating mouse ESCs into a variety of blood cell types, though he admits that generating lymphocytes is still a problem. His lab has developed the requisite protocols by trial and error over the past decade.19 When removed from conditions that keep them in an undifferentiated state, ESCs form clusters of differentiating cells called embryoid bodies. "At that point, we take the cells from the embryoid body and put them into cultures containing cytokines that stimulate the growth and maturation of blood-cell progenitors," Keller recounts. "Alternatively, we can first isolate the blood-cell progenitors from the embryoid bodies by using antibodies to specific cell-surface markers and then put them into culture." Keller is now searching within embryoid bodies for the hematopoietic stem cell equivalent to the hematopoietic ASC that other labs have isolated alternative hypotheses that could help explain transdifferentiation. One theory is that ASCs in various organs all originate from ASCs in bone marrow; these ASCs then adopt organspecific traits after being seeded in local environments. The other theory is that ASCs arise independently in various organs but share phenotypic and functional characteristics. Thus, ASCs from one organ can generate mature cells of another organ because the ASCs of both organs have a common origin and/or exhibit certain common features. In a 1999 Nature paper, a team headed by Mulligan and his Harvard colleague, Louis M. Kunkel, reported that injecting muscle-derived ASCs into irradiated mice led to reconstitution of the recipients' hematopoietic compartment.1 ASCs with this capability were designated muscle SP ("side population") cells. Like hematopoietic SP cells, muscle SP cells resisted staining by a Hoechst dye. The two SP cell types weren't identical, however. In unpublished work since then, "we've in bone marrow. This putative stem cell in the embryoid body has been harder to find, he says, because it "appears to be more immature than the one in adult bone marrow." His approach is to transplant candidate stem cells into mice with drug-damaged hematopoietic systems and then to observe whether blood-cell re-population occurs. When might his methods boost human blood supplies for transfusions? "Some years away" is all that Keller will predict. Douglas Steinberg is a freelance writer in New York. References 1. D. Steinberg, "Stem cell discoveries stir debate," The Scientist, 14:1,14-5, Nov. 13, 2000. 2. S.-H. Lee et al., "Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells," Nature Biotechnology, 18:675-9, June 2000. 3. L. Studer et al., "Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats," Nature Neuroscience, 1:290-5, 1998. 4. L. Studer et al., "Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen," Journal of Neuroscience, 20:7377-83, Oct. 1, 2000. 5. J. Wagner et al., "Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes," Nature Biotechnology, 17:653-9, 1999. 6. C.B. Johansson et al., "Identification of a neural stem cell in the adult mammalian central nervous system," Cell, 96:25-34, 1999. 7. F. Doetsch et al., "Subventricular zone astrocytes are neural stem cells in the adult mammalian brain," Cell, 97:703-16, 1999. 8. N. Wade, "Brain stem cell is discovered, twice," New York Times, p. F3, June 15, 1999. 9. One paper is in press. For the other, see J.C. Conover et al., "Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone," Nature Neuroscience, 3:1091-7, November 2000. 10. D.L. Clarke et al., "Generalized potential of adult neural stem cells," Science, 288:1660-3, June 2, 2000. 11. B.J. Chiasson et al., "Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics," Journal of Neuroscience, 19:4462-71, 1999. 12. E.D. Laywell et al, "Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain," Proceedings of the National Academy of Sciences (PNAS), in press. 13. B. Soria et al., "Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice," Diabetes, 49:157-62, February 2000. 14. V.K. Ramiya et al., "Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells," Nature Medicine, 6:278-82, March 2000. 15. S. Bonner-Weir et al., "In vitro cultivation of human islets from expanded ductal tissue," PNAS, 97:7999-8004, July 5, 2000. 16. N.D. Theise et al., "Liver from bone marrow in humans," Hepatology, 32:11-6, July 2000. 17. E. Lagasse et al., "Purified hematopoietic stem cells can differentiate into hepatocytes in vivo," Nature Medicine, 6:1229-34, November 2000. 18. M.G. Klug et al., "Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts," Journal of Clinical Investigation, 98:216-24, 1996. 19. See, e.g., M. Kennedy et al., "A common precursor for primitive erythropoiesis and definitive haematopoiesis," Nature, 386:488-93, 1997. Precursor Cells to the Rescue? Two reports out this month suggest that less-than-fully differentiated cells-whether embryonic or adult--could help humans recover from a host of nervous system ailments, ranging from motor neuron diseases to brain cancer. In a study announced at the Society for Neuroscience meeting in New Orleans Nov. 4-9, researchers from Johns Hopkins University School of Medicine Left: Immunohistological staining for epithelialspecific cytokeratins of a female liver allograft in a male recipient. Right: Fluorescent in situ hybridization for X and Y chromosomes. and Harvard Medical School infected rodents with Sindbis virus, which causes limb paralysis by attacking the motor neurons that thread from the spinal cord to the muscles. The team then injected embryonic germ cells (EGCs), primordial cells that are pluripotent like stem cells, into the cerebrospinal fluid (CSF) at the base of the animals' spines. The EGCs were pretreated with growth factors to nudge them onto the path of neural differentiation, thereby preventing them from developing into tumors. Within several weeks, the EGCs had migrated to the ventral horn of the spinal cord, which contains the cell bodies of motor neurons. And by eight weeks after injection, 11 out of 18 animals had regained the ability to place the soles of one or both of their hind feet on the ground. Yet, only about 6 percent of the migrating EGCs seemed to have differentiated into neurons, as indicated by expression of cell-surface markers. Earlier animal studies showed that applying stem cells to the site of a traumatic spinal-cord injury leads to some functional recovery. The newly reported experiments were the first involving a diffuse disease that affects the whole spinal cord and the first in which primordial cells were delivered via CSF, according to lead researcher Douglas A. Kerr, an assistant professor of neurology at Hopkins. In ongoing work, "We're trying to characterize why the animals recovered," he says. "And we're also trying to trick the cells prior to implantation into really thinking that they are to be motor neurons. Presumably then we might even see a better functional recovery." Before Kerr and his colleagues turned to EGCs, their study used human neural stem cells (NSCs) derived from a fetus' telencephalon by Evan Y. Snyder, an assistant professor of neurology at Harvard Medical School.1 Kerr recalls that these NSCs restored some function to a handful of rodents, but that they showed no effect in later experiments on a larger group of animals. Snyder and a Harvard team, meanwhile, used the same NSCs in a newly published study on brain cancer.2 After the researchers implanted glioblastoma cells into rodents, the animals developed intracranial tumors. NSCs, which were implanted several days later, infiltrated and surrounded the tumors, and chased down malignant cells that were migrating into normal tissue. Tumor targeting occurred even when the NSCs were introduced far from a tumor, such as into the vein of an animal's tail. When NSCs expressed cytosine deaminase, an enzyme that converts a non-toxic pro-drug into a chemotherapeutic agent, one mouse's tumor shrank about 80 percent. The researchers found that the NSCs neither differentiated nor turned tumorigenic in the recipient rodents. One message of the study, says Snyder, is that "if there's pathology, not only can there be very dramatic, extensive [NSC] migration, but it happens along nonstereotypical, unpredicted pathways." Why do NSCs track brain tumor cells? He suggests some possibilities: Some oncologists view brain-tumor cells as NSCs "gone bad," and these two similar cell types might respond to the same cues. In addition--or alternatively--tumors or the brain cells that they're killing might secrete factors that attract stem cells. Snyder and his colleagues have been holding talks with the Food and Drug Administration about using NSCs as adjunctive therapy to treat brain tumors, which are now almost always incurable. He notes that NSCs could be equipped with transgenes that fight cancer by promoting differentiation or blocking angiogenesis. --Douglas Steinberg References 1. J.D. Flax et al., "Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes," Nature Biotechnology, 16:1033-9, 1998. 2. K.S. Aboody et al., "Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas," Proceedings of the National Academy of Sciences, 97:12846-51, Nov. 7, 2000. Tissue Engineering Illustrated below is one approach to tissue engineering. To create a living replacement tissue, a small number of cells can ideally be harvested from the patient using a biopsy and then cultured to the appropriate numbers in the laboratory. These cells can then be grown within a three-dimensional natural or synthetic scaffold and in the presence of appropriate growth and differentiation factors. If provided with the appropriate conditions and signals, the cells will secrete various matrix materials to create an actual living tissue that can be used as a replacement tissue to be implanted back into the defective site in the patient. The scaffold should ultimately degrade to prevent certain risks that can occur with the long-term presence of any foreign material in the body. If cells from the patient are used, then there will be no immune rejection response to the implanted tissue.