SCIENCE & TECHNOLOGY STEM CELL SUPPORT Chemical engineers de Pablo (left) and Palecek are working to make the maintenance and growth of stem cells easier.
STEM CELL CHEMISTRY Chemists and engineers offer help in identifying molecules that direct stem cell behavior LOUISA WRAY DALT0N, C&EN WASHINGTON
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speak of the great promise of the stem cell. An embryonic stem cell, she might say has the capability to become anything from ablood cell to abrain cell and therefore has the potential to replace all types of failing tissue. We hope, she might say to eventually use the stem cell as medical therapy Chemists or chemical engineers who foray into the field of stem cell research take a different perspective. Stem cells require the right environment to receive the right signals to turn into the desired cells. Small molecules that can manipulate the stem cell would therefore also be powerful in medical therapy, perhaps even more powerful and convenient than the stem cell itself. By concentrating on what and how small molecules can help the stem cell grow and differentiate, chemists and chemical engineers are poised to contribute uniquely to the development of the field. "We dunk," says Sheng Ding, a chemist at Scripps Research Institute, "if you understand what the signals are that control stem cell fate—and we know there are stem cells reserved in pretty much all the tissues in adults—we can possibly develop small-
molecule therapeutics or other types of therapeutics that stimulate in vivo regeneration. That {for us} would be the ultimate goal of stem cell research. We think those therapeutics will be the future of regenerative medicine, not just the stem cell." Although the therapy Ding proposes is a step beyond the traditional view of stem cell therapy, it is a natural concept for a chemist. His goal is a small molecule. Ding and collaborator Peter G. Schultz, also at Scripps, are two of relatively few chemists now working with stem cells. They are, however, joined by biologists, geneticists, and others in emphasizing the importance of the small molecule in stem cell research. "Controlling stem cell fate is challenging," Ding says. Everyone wants to identify the signals that direct stem cell behavior, but the signaling pathways turned on in stem cells are still being tracked down. Developmental biologists, fortunately, have made great strides in laying the foundations of the signaling pathways that are important in both frog egg and human embryo development. With those pathways, stem cell scientists are starting to elucidate the chemistry and biology of stem cells. The term "stem cell" encompasses sev-
eral kinds of cells. The human embryonic stem cell is the most magical of all, because it still has the promise ofbecoming any type of cell in the human body It is also the focus of heated ethical debates, because using it for research or therapy means that a once-viable human embryo will not thrive. Many researchers also work with mouse embryonic stem cells, which are more readily available and easier to manipulate. Other types of adult stem cells—in humans and other organisms—have a more limited potential for differentiation but still retain some "sternness." For example, blood stem cells that reside within human adult bone marrow can reconstitute the entire blood system of the body Depending on the tissue, however, adult stem cells can be hard to find. Human heart tissue, for example, was only recendy found to be harboring adult stem cells. And finding stem cells isn't the only challenge. Growing them outside the body and directing them toward particular cell lineages are further hurdles. With any type of stem cell, understanding its signaling pathways is key to deterrnining its fate. DING AND SCHULTZ are taking a discovery approach to finding new small molecules that can direct the fate of stem cells. They are searching for molecules that induce stem cells toward a specific lineage, keep the cells undifferentiated, or take differentiated cells back to a stem cell state. To do so, they have created combinatorial libraries based on molecular motifs known to be involved in common signaling pathways. Because no one knows which signaling pathways are important in stem cell regulation, they have made their libraries as diverse and unbiased as possible. They then treat stem cells in culture with molecules from the libraries. The Scripps group has found a number ofactive molecules this way including a 4,6disubstituted pyrrolopyrimidine that directs mouse embryonic stem cells to become nerve cells [Proc. Natl Acad. Sci USA, 100,7632 (2003)}. Ding and Schultz subsequendy identified one of this molecule's cellular targets and the pathway that it activates: It tighdy binds to glycogen synthase k i n a s e ^ , an important member ofthe sig-
Small molecules that can manipulate the stem cell would also be powerful in medical therapy. HTTP://WWW.CEN-ONLINE.ORG
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SCIENCE & TECHNOLOGY same developmental pathways that work correcdy in stem cells often work incorrecdy in cancer cells. One chemical engineer has taken advantage ofthe similarities between cancer cells and stem cells by applying a nov el biochemical tool developed for cancer cells to stem cell research. JUST A HANDFUL ofpathways—the same ones that are important in developmental Stem cell cultures, like cancer cell cul biology—likely play the leading roles in di tures, are rarely pure, saysJulie Audet, an as recting stem cell fate. Wnt is one pathway sistant professor at the Institute of BiomaHedgehog, BMPATGF-β, Notch, and FGF terials & Biomedical Engineering at the are others. Generally in combination, these University ofToronto. Cancer cell cultures signaling pathways control the patterning constandy accumulate mutations, and stem and growth of early embryos. cells constandy tend toward differentiation. Performing a traditional biochemical as By starting with these pathways, the re say requires a large population of (ideally) search group of Mickie Bhatia, director of identical cells. When a heterogeneous pop stem cell biology and regenerative medicine ulation is used, measurements on all the at Robarts Research Institute, in Ontario, cells—pure stem cells and differentiated has been trying to identify cellular factors cells—are averaged. That means "you don't important in differentiation. Bhatia works have good information about what is go primarily with blood-forming stem cells, ing on in the stem cells," Audet says. which are found in adult bone marrow and newborn umbilical cord blood. Cord blood For her postdoctoral research, Audet is the most enriched source of these cells. looked into ways of measuring what is hap Even that source, however, is not enough pening in a single cell. She found an assay at for one adult bone marrow transplant be the University of California, Irvine, in the cause the total volume in cord laboratory of cancer researcher blood is small. One of Bhatia's Nancy L. Allbritton. The assay major goals is to discover which consists offirstloading a cell with signaling factors can help re enzyme reporters—fluorescent searchers grow blood-forming peptides designed to be modified stem cells in culture for thera by only one cellular enzyme each. peutic use. Once inside, the reporters are modified by their respective en Bhatia and his group knew zymes, the cell is lysed, and the from developmental biologists contents of the cell are then sep that blood cells develop out of arated and quantified by capillary an embryonic germ layer called NERVE DIRECTOR Mouse embryonic stem cells treated electrophoresis. the mesoderm. They hypothe with a molecule (structure shown) that induces neuronal sized, says Krysta Levac, a re differentiation light up when injected with two fluorescent The results indicate the level search associate in Bhatia's lab antibodies (red and green) that bind to neuronal markers. of activity of each targeted en oratory, that the signaling zyme in just one cell, instead of factors involved in directing mesoderm averaging the characteristics of a heteroge tivators of the Hedgehog pathway have cells to become blood tissue might help neous population. Another "advantage of shown potential in treating neurodegener human blood-forming cells grow in a petri this approach is that you can monitor the ative disease, and Curis has partnered with dish. activity ofseveral enzymes at the same time," "Wyeth Pharmaceuticals to develop them. Audet says. At the moment, she is develop In addition, some inhibitors of the Hedge It turns out they do. Sonic Hedgehog, ing reporters for each enzyme in the Notch a protein ligand in the Hedgehog path hog pathway may effectively treat basal cell and Wnt signaling pathways. carcinoma and solid tumors. Curis is work way; Bone Morphogenetic Protein-4, a ing with Genentech on this project. ligand of the BMP pathway; and at least two ligands in the Notch pathway, JaggedRubin says that influencing a fundamen THE LACK OF pure stem cell populations 1 and Delta-1, help blood-forming stem tal pathway like Hedgehog, which coordi for research is not the only challenge of cells survive and proliferate in culture. working with stem cells. Basic culture and nates the activity of many other pathways, Bhatia and coworkers found that a mem maintenance of stem cell lines is demand leads the body to respond in multiple ways. ber of the Wnt pathway Wnt-5A, although ing and tedious. Another team of chemical The complexity of the response can be ineffective in culture, enhances the abili engineers is working on just this problem. daunting, but it also means Curis' drugs have ty of human blood stem cells to repopuHuman embryonic stem cells "grow very the potential to effect multiple therapeutic late mouse blood in vivo. slowly and our ability to preserve them is responses. For example, Cutis' Hedgehog poor," says Sean P. Palecek, a chemical en inhibitors both directly block tumor cell Such results highlight the mteitwining gineer at the University of Wisconsin, proliferation and cut off the blood supply ofmultiple pathways in regulating stem cell Madison. Few cells survive the suspension, to tumors by inhibiting the production of fate. In the past, Levac says, 'Sve've looked freezing, thawing, and replating that occur growth and angiogenic factors. at pathways one at a time—in isolation. But during cryopreservation, the method used we are now working on looking at them in Using stem cells to look for cancer ther combination." If BMP-4 increases the life apies, as Cutis has done, is logical because the to preserve almost all types of cells. Ά ρ naling pathway called Wnt. The Scripps chemical library screen thus identified not only a molecule that regulates stem cell fate but also at least one of the pathways im portant in neuronal differentiation.
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time of stem cells andjagged-l induces them to proliferate, "it is quite possible that if we add the molecules together, we may end up with better effects than if we added them one at a time," Levac says. It is not surprising that pathways active in stem cells often work in tandem. 'Alot of these pathways regulate really fundamental processes," says Lee L. Rubin, chief scientific officer at Cutis, a biotechnology company cofounded by Harvard University stem cell researcher Douglas A. Melton. For exam ple, the Hedgehog pathway "controls the balance between proliferation and differ entiation. Basically it regulates how many cells you have and what kinds they are." That's why the three ligands of the Hedge hog pathway "are involved in pretty much the development ofevery tissue," Rubin says. Curis was founded to explore how study ing embryonic development in stem cells could lead to new disease therapies. Cutis has specifically focused on identifying and de veloping drug candidates that either inhibit or activate the Hedgehog pathway Cutis has some promising leads. A few ac
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proximately 0.1% of the cells will survive these processes," Palecek says. In addition, the stress ofthe freezing-thawing cycle promotes uncontrolled differentiation. Palecek and his collaborator Juan J. de Pablo, also a chemical engineer at Wisconsin, are investigating not only the chemical factors but also the physical stresses F U N D I N G
that affect growth and preservation of human embryonic stem cells. For example, freezing a stem cell can create osmotic stresses related to the changing concentration of solutes as ice forms. There's also the thermal stress of freezing. Finally, cells can be stressed by the addition of cryoprotectants such as dimethyl sulfoxide
UPDATE
States Are Stepping Up To Fill Funding Void Left By Restrictive Federal Policy esearch on embryonic stem cells has the potential to elucidate treatments and cures for a variety of diseases. Although work in this area is at an early stage, researchers are beginning to understand more about how these cells work and are striving to push in new directions. An important factor that will limit the rate at which researchers proceed is funding. Currently, federal funding is limited to work that involves cell lines derived before Aug. 9, 2001 (C&EN, July 19, 2004, page 16). This means that researchers wanting to work with cell lines derived since that time must find funding from sources outside the federal government. One funding source that is increasing its efforts to fill the void left by the federal government is state legislatures. Most notable has been California, which included an initiative, called Proposition 71, in the ballot for the November 2004 elections. Proposition 71 got the thumbs-up from voters to provide $3 billion for embryonic stem cell research over 10 years through the sale of bonds. The measure also creates the California Institute for Regenerative Medicine and will begin funding research later this year. With this action, California has set itself apart. It hopes to pull talented scientists interested in doing stem cell research away from other states and into its research institutions and facilities. The threat of losing talent has prompted other states to take action. "Every research-intensive state is concerned about the competitiveness issue that they are going to face in the areas of stem cell research and, more broadly, biology-based research because of the investment California is going to make," says Lawrence Solar, vice president for government relations at the Juvenile Diabetes Research Center. JDRC is an ac-
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tive supporter of and a funding source for stem cell research. Solar notes that at least 10 states are considering legislative initiatives akin to California's. For example, Illinois legislators are drafting a proposal to increase the state's support of stem cell research by providing $1 billion for grants over a 10-year period, subject to approval in a statewide referendum. Legislators from
FOCUSING Biologist studies culture trays containing human embryonic stem cells. New York have also proposed a $1 billion stem cell initiative funded by bond sales. Elsewhere, New Jersey Acting Gov. Richard J. Codey (D) has called for a $380 million investment in stem cell research, and Wisconsin Gov. James E. Doyle (D) has pledged $750 million in support. Other states, including Connecticut, Maryland, and Virginia, also have pledged money for stem cell research. "The passage of the initiative in California has really changed the landscape quite a bit," Solar says, adding that the number of states passing measures to fund stem cell research will continue to grow. "I think there is no doubt about it." That's good news for researchers who can't afford to put parts of their research on hold until the federal policy changes. -SUSAN MORRISSEY
(DMSO). Combinations of these stresses cause many cells to undergo cell suicide— or apoptosis—during ciyopreservation. De Pablo and Palecek are looking for ways to "maintain the antiapoptotic signals" that are present before preservation. They have tried using the disaccharide trehalose as a cryoprotectant in addition to DMSO. DMSO is a common cryoprotectant for mammalian cells because it can cross the cell membrane and lessen some of the osmotic stress experienced by the cell during freezing. However, DMSO is toxic to cells. Trehalose, on the other hand, is a natural cryoprotectant used by yeast and other organisms for protection during freezing or drying. Unfortunately trehalose on its own cannot cross the cell membrane. Palecek and de Pablo are now trying different methods of loading the cells with trehalose. They have also embedded the cells in an extracellular matrix called Matrigel before freezing. (Most researchers grow human embryonic stem cells on a layer of mouse fibroblast feeder cells.) The Wisconsin researchers have found that the combination of applying trehalose and embedding the cells in Matrigel raises cell viability from 0.1% to about 10%—"more in line with what we would need to ease the cryopreservation viability problem," Palecek says. Matrigel is extracted from a mouse tumor. The mouse cell environment works well because it contains factors that support the growth ofhuman embryonic stem cells without inducing differentiation. Nevertheless, de Pablo stresses that developing a purely synthetic matrix would be much better. As the popular media have recently highlighted, the current practice of growing human embryonic cells in an environment containing animal derivatives will be problematic ifthe cells or their progeny are ever going to be transplanted into humans for therapeutic purposes. Creating a synthetic matrix on which to growhuman embryonic stem cells is just one ofthe chemistry-related challenges that face the stem cell research community Finding the factors that such a matrix requires will take the collected effort ofmany types ofresearchers,fromchemical engineers like Palecek and de Pablo to biologists like Bhatia. Stem cell research has benefited from advances in developmental biology in elucidating the biochemical pathways that are active in stem cells. Now scientists are just beginning to learn how those pathways and the factors in them interact to determine the behavior of stem cells. "Better chemistry and better materials," de Pablo says, "are going to be crucial for the future of stem-cell-based therapies." • C & E N / FEBRUARY U ,
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