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NANOTECHNOLOGY Nano and Stem Cells: Crossroads Technologies Mapped at Korea Conference Two of the most promising and controversial areas of research, nanotechnology and stem cells, shared the stage at a June symposium in Seoul, Korea, showing international progress and directions for the near future. The Fourth Asan-Harvard Joint International Symposium, titled "Nanotechnology in Biology and Medicine," took place on the 15th and 16th, coincidentally just a week after Eric Drexler, considered the father of nanotechnology, disavowed a prediction he had made about the field, that runaway nanoscale self-replicators, or "grey goo," could threaten society. Nanotech enables intervention on the level at which biological systems actually operate. Reflecting the new optimism, the conference focused on promising areas of drug design and delivery, nano-based diagnostics and therapies, tissue engineering, "lab-on-a-chip" devices, and genomics. Hosted at Asan Medical Center and organized jointly by Asan and Harvard Medical School's Harvard Medical International (HMI), the symposium drew experts from Korea and the United States. In her keynote address, Lynn Jelinski, a chemist, former Cornell professor, and now president of Sunshine Consultants International in Florida, said that nanotechnology along with genomics "is definitely going to change the quality of the human condition." One of its attractions is that nanoscale clusters of molecules have different physical properties, as in conductivity, strength, and optical transport, than the same material on a bulk scale. So it is possible to develop novel circuits and to find new uses for familiar substances. Another advantage, she said, is that "biological systems have very well-defined rules of self-assembly," and these can be harnessed for predetermined purposes. Jelinski explained that in 2000, the World Technology Evaluation Center (sponsored by multiple federal research agencies) projected that the worldwide impact of nanotechnology by 2015 would top a trillion dollars per year. But gaps in our knowledge remain. "Do we understand the toxicity of nanostructures?" Jelinski asked. Not entirely. She said it is up to scientists to examine the real dangers and to develop public understanding of the technology to avoid a backlash similar to that against genetically modified food and other organisms. Framing the situation of nanotechnology vis-à-vis the public, presenter Mehmet Toner, professor of biomedical engineering at Massachusetts General Hospital (MGH) and Harvard Medical School and director of the Microsystems Bioengineering Laboratory at MGH, said that nanotech seems to have a relative lack of support and understanding in the public mind. Unlike the space race of the 1960s, he said, nanotech has no moon landing to shoot for. Unlike stem cell research, it has no prospect for an Alzheimer's cure. Further confusion surrounds the prefix nano. Strictly speaking, nano refers to billionths of a meter, and nanotechnology applies to methods of manipulating objects 100 nanometers or smaller in size. But few people speak strictly about nanotechnology-Toner even changed one of his talk titles to say "tiny" instead of "nano." For practical purposes, in biomedical engineering, the micro (millionth) scale blends with the nano scale. What is important is that nanotechnology enables intervention on the scale at which biological systems actually operate. Tissue Engineering One of the aspects of nanotech that Toner addressed was the impact of nanosystems on tissue engineering. The field makes use of microfabrication techniques to produce functional tissue in both two and three dimensions. Either template-directed self-assembly of the living tissue or spontaneous self-assembly may be used, depending on the application. Toner described studies in which tiny engineered surfaces, some with islands of binding or nonbinding molecules, control cell shape and differentiation. "By precisely controlling the shape and the type of the extracellular matrix of cells using micropatterned islands of cell adhesive and nonadhesive molecules, it is now possible to build three-dimensional tissue equivalents for regenerative medicine," he said. Evidence also shows that nanopatterned surfaces influence the adhesion and growth of cells. This work makes use of previous findings that if cells can spread a great deal, they proliferate, and if they cannot spread enough, they die by programmed cell death. "Cell shape can also regulate stem cell lineage commitment," Toner said, suggesting an area where nanotech and stem cell research may converge for generating certain adult cells for medical applications. In a complementary presentation on cellular responses to nanopatterned polymeric surfaces, Kyu-Back Lee, assistant professor of biomedical engineering at the College of Medicine, Korea University, said that some of the minutely corrugated and porous templates he is developing may be used in the future for embryonic stem cell proliferation and guidance of cell differentiation. Toner also reviewed the nano research with blood cells that he and his colleagues are conducting at the BioMEMS Resource Center at MGH. (MEMS is shorthand for micro-electromechanical systems.) The blood is, in effect, the body's health sensor, he said, and microfluidic systems for separating various cells from the blood can vastly speed up analysis. To get a pure population of leukocytes for research, for example, one question is how do you get rid of the red blood cells? Toner showed a video of a microfluidic system that he and his colleagues developed that reduces separation time from about a half hour to only a few seconds. Small, portable lab-on-a-chip and MEMS devices can also combine molecular, cellular, and fluidic analyses for applications like diagnosis at the point of care and work in drug discovery. Though Toner argued that such devices are still looking for major applications, they could potentially be used for early diagnosis of cancer and infectious and immune diseases. "In the case of therapeutics, it's probably stem cells and dendritic cells [that are the likely targets of these technologies], finding those cells and repopulating them and giving them back to the patient for various applications," he said. Since lab-on-a-chip and MEMS techniques are well established, reliable, and economical, the chips are amenable to use in poorer countries. The devices can be made for cents, Toner said, and they are still very accurate. Lab-on-a-Chip As the CEO of Digital Bio Technology in Korea, Jun-Keun Chang is developing lab-on-a-chip and other devices for world markets. Also an assistant professor of electrical engineering and computer science at Seoul National University, Chang presented at the conference, describing fluidics-based devices that enable single-molecule and single-cell manipulation and detection. "We can look into multiple events within living cells simultaneously by real-time monitoring and fluorescence measurement at the single-cell level," he said. Among the clinical applications he mentioned was that lab-on-a-chip devices like his could potentially be used prenatally, replacing amniocentesis with a noninvasive blood test. One clinical goal Chang has is to be able to isolate rare cells such as stem cells. A useful tool would be his micro-FACS (fluorescence-activated cell sorter), which reduces the conventional desk-sized FACS to a hand-held instrument while maintaining relatively high throughput, up to 1,000 cells per second. Though his products face a lot of competition, he explained, his cell sorter is particularly versatile and may be adapted to a variety of target cell types. Like Chang, Toner is investigating single-cell events in a controlled microenvironment. "We would like to clearly understand the systems biology of a single cell," Toner said. "We would like to know every single gene that's expressed in that cell. We would like to know every protein in that cell and every surface antigen." Toner also would like to understand the dynamics of gene expression within a cell. Of particular interest are cancer cells, stem cells, and dendritic and other immune cells. Techniques such as photolithography, etching methods, and nanoengineering can produce many tiny, purposeful features on silicon and glass substrates. These include nanoscale biochemical reaction chambers, bioseparation channels, arrays of biological molecules, microelectronic components, and tiny pumps and valves to regulate fluid flow. These techniques come to play in creating the closed-system devices that enable single-cell investigation. Toner described one such device he and his colleagues developed, a single-cell lysis system, to dissolve cellular membranes in order to access and analyze target molecules within, thereby building an understanding of single-cell behaviors. Referring to the fusion of this kind of biotechnology and information technology as "bio-IT," Han-Do Kim, professor of molecular biology at Pusan National University and director of the Asian Institute of NanoBioScience and Technology, said that the field was dominated by the United States and relatively underdeveloped in Korea. Yet he argued that the emerging industry "is particularly suitable for Korea to foster since we have intellectual ability in application in a variety of fields as well as the world's best IT infrastructure and human resources, including excellent semiconductor design and manufacturing." Among the initiatives under the Korean bio-IT umbrella are those in chemical genetics and genomics conducted by Tae-Kook Kim, associate professor of biological sciences at the Korea Advanced Institute of Science and Technology. He and his colleagues are using small molecules to probe and control the function of specific pathways and networks in human cells. They are applying these approaches to uncover pathways involved in cancer and neurodegenerative diseases. Information Validity But all of these tiny-tech instruments and approaches generate data, and in many cases, methods for gleaning reliable information from it prove to be inadequate. Peter Park, instructor in pediatrics at Children's Hospital, Boston and associate director of bioinformatics at the Harvard Medical School-Partners Center for Genetics and Genomics, is devising statistical and computational methods for interpreting data from a variety of high-throughput technologies like microarrays, genomic sequences, and protein-protein interactions. One of his interests is finding relationships between genomic data from microarrays and patient data, in order to develop methods for predicting patient survival based on molecular profiles. Microarray studies still have bugs, Park said, which prevent them from being used for clinical care. A major problem is a lack of agreement among studies for the same disease. He cited several examples in which different technological platforms, analysis techniques, or patient populations produced widely varying results in terms of genes identified and predictive performance in classifying patients. Especially for complex diseases, microarrays have often failed to reproduce results reported in the scientific literature, Park said. There also is a push toward using proteomic data for early diagnosis by detecting small changes in the abundance of certain proteins present in the patient. Park reported, however, that a careful re-analysis of a widely publicized study appears to show that the normal and cancer patients were distinguished not by differences in their underlying biology but by artifacts in the generation and processing of the data. "Microarrays will enable personalized medicine as we all had hoped," he said, "but it will require more reproducibility and careful validation as well as improvements in bioinformatics before they can be used in practice." Drug Delivery One particularly promising area of nanotechnology is drug delivery. Nanotech devices for delivering drugs have numerous advantages over their macroscale counterparts, said Nicholas Peppas, the Fletcher S. Pratt chair of engineering at the University of Texas, Austin. He referred to an investigation he was involved in that was being announced at another conference on the same day. He said it was the first successful in vitro study of oral delivery of interferon-beta 1a, used for multiple sclerosis. He knows well what this may mean for patients. "I will not leave you in this audience this morning and tomorrow afternoon without pointing out to you how important quality of life is," Peppas said. He explained that patients take the drug interferon-beta. It works well except that it must be taken by intramuscular injection once a week; so at first, it has a very high concentration in the blood, causing weakness and pain. "The first 24 hours after it's taken," he said, "patients are in hell." The study tested oral delivery of peptides and proteins, including interferon-beta 1a, through a hydrogel carrier. Engineered on a nano scale and composed of crosslinked polymers, the pH-sensitive hydrogels incorporated the drug and then released it at variable rates, depending on the pH of the surrounding environment. The research suggests that these nanoparticle carriers may protect drugs from being broken down in the body until they can reach the small intestines. Animal studies are just beginning. Underscoring the promise of engineered technologies to deliver medications, Sang-Yoon Kim, professor of otolaryngology at Asan Medical Center and its teaching affiliate, the University of Ulsan College of Medicine, presented his work on microspheres loaded with all-trans-retinoic acid (atRA) to treat head and neck squamous cell carcinoma, an aggressive epithelial malignancy. Clinical application of atRA is limited due to retinoid resistance and toxicity. His team's study showed that unlike daily oral doses, biodegradable microspheres that were injected under the skin maintained plasma concentration of the drug in the therapeutic range for a long period. Looking into the future, Peppas said that these and other technologies may be used to construct smart diagnostic and therapeutic systems. He visualizes giving people an injection containing microparticles or nanospheres, which have been imprinted to recognize disease-associated compounds like glucose, angiotensin, and cholesterol. The "stealth" particles would circulate in the blood, and when they recognized an enemy compound, would capture it, and through biodegradation, trigger the release of an appropriate therapeutic drug. Stem Cell Controls In his work on the natural systems that regulate adult hematopoietic stem cells, David Scadden, professor of medicine at MGH and HMS, codirector of the Harvard Stem Cell Institute, and director of the Center for Regenerative Medicine and Technology at MGH, has identified for the first time elements of the microenvironment that control the behavior of mammalian stem cells. He and his colleagues discovered that mineral components are important to stem cell localization; matrix components are important to constraint of stem cells; and bone-forming osteoblasts, to the support and proliferation of stem cells. More specifically, Scadden commented that the calcium-sensing receptor, located on the surface of hematopoietic stem cells and other cells, is critical to stem cells finding their niche. If stem cells are in an environment of low ionic calcium, the receptor is downregulated. "And as you may know," Scadden said, "in the blood bank, that's exactly the way we store stem cells. So, in fact, our method of storage may be working against us in terms of the ability to get these cells to engraft. So we are now working with the blood bankers to see if methods of storage can be used which could possibly change this expression and enhance engraftment." Scadden said that his previous work on stem cell control suggests that the cyclin dependent kinase inhibitor p21, identified as being important for regulating the hematopoietic stem cell, may influence other adult stem cell types as well. His team looked at the nervous system in a stroke injury model and found evidence that reducing p21 might enhance regeneration of nerve cells after injury. In a paper in the May Nature Cell Biology, his team reports that decreasing another cell cycle inhibitor, p18INK4C, also increases the stem cell pool. A general goal of the above work is to understand the difference between embryonic stem cells and adult stem cells in proliferation control. Embryonic stem cells, which unlike adult stem cells can regenerate any tissue in the body, can be maintained in culture almost indefinitely. They proliferate robustly, having no boundary between the G1 and S phases of the cell cycle as do adult stem cells. But the adult somatic stem cell has a profound G1-S blockade, Scadden said, and understanding the difference might make adult stem cells more useful in therapies. Presenter Mi-Jung Kim, an assistant professor of laboratory medicine at the Asan Institute for Life Sciences, University of Ulsan College of Medicine, also is investigating hematopoietic stem cells to improve their therapeutic value. Her talk was introduced by stem cell researcher Shin-Yong Moon, director of the national Stem Cell Research Center and of the Institute of Reproductive Medicine and Population, both at Seoul National University, who was a coleader of the team that in February first reported developing pluripotent human embryonic stem cells from a cloned human blastocyst; embryonic stem cells previously had been produced with cells from mice using the same method, somatic cell nuclear transfer. Kim has conducted recent studies on the integration and proliferation of transplanted bone marrow cells in the recipient's body. Many studies have suggested that there is an inverse relationship between cell cycle activity and this process of engraftment. Findings have also shown connections between engraftment and cytokine receptors such as CXCR4, the receptor for the chemokine stromal derived factor-1a (SDF-1a). Research also suggests that SDF-1a is a key regulator of stem cell migration and homing and that pluripotent human stem cells express its receptor, CXCR4. Using the fluorescent dye rhodamine-123 (Rh-123) to segregate mouse hematopoietic stem and progenitor cells into two distinct subsets with different homing potentials, the Rh-123-low stem cell population and Rh-123-high progenitor population, Kim and her colleagues showed that cell cycle progression by itself cannot account for the decrease in potential for repopulation observed after ex vivo expansion. "Our data suggest that the homing potential difference between Rh-high and Rh-low cell populations could be explained by their membrane CXCR4 expression level rather than their cell cycle status," Kim explained. The Nanotechnology symposium was opened by Kun-Choon Park, president of Asan Medical Center and professor of surgery at the University of Ulsan College of Medicine, who said that nanotechnology "can lead via improved biodevices to a better quality of life, improved wealth creation, and a stronger base for our new knowledge-based economy." He also thanked the cochairs of the symposium organizing committee, In-Koo Kim, vice president of education and research at the University of Ulsan College of Medicine and Asan Medical Center, and Mitchell Spellman, director of academic alliances and international exchange programs at HMI. In his greeting, Robert Crone, president and CEO of HMI, pointed out that since each Asan-HMI symposium has been multicultural and multidisciplinary, "each event has been a learning experience for the faculty as well as the audience, enabling even those who are experienced in their subject to look at their research from a different perspective." In closing the conference, which included a poster session and award ceremony for the top three posters, Spellman described a paradigm shift put in motion by work at the nano scale, the level at which biological systems function: "These lectures and the promise and the power of nanotechnology," he said, "have already or will very soon obliterate or obscure the distinction between what we've called pure science and applied science." --Robert Neal