Stem Cell Chemistry

A stem cell biologist will speak of the great promise of the stem cell. An embryonic stem cell, she might say, has the capability to become anything from a blood cell to a brain 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 think," 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 several kinds of cells. The human embryonic stem cell is the most magical of all, because it still has the promise of becoming 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 "stemness." 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 recently 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 determining 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 of active molecules this way, including a 4,6-disubstituted pyrrolopyrimidine that directs mouse embryonic stem cells to become nerve cells [Proc. Natl. Acad. Sci. USA, 100, 7632 (2003)]. Ding and Schultz subsequently identified one of this molecule's cellular targets and the pathway that it activates: It tightly binds to glycogen synthase kinase-3b, an important member of the signaling 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 important in neuronal differentiation.

JUST A HANDFUL of pathways--the same ones that are important in developmental biology--likely play the leading roles in directing stem cell fate. Wnt is one pathway. Hedgehog, BMP/TGF-b, Notch, and FGF are others. Generally in combination, these signaling pathways control the patterning and growth of early embryos.

By starting with these pathways, the research group of Mickie Bhatia, director of stem cell biology and regenerative medicine at Robarts Research Institute, in Ontario, has been trying to identify cellular factors important in differentiation. Bhatia works primarily with blood-forming stem cells, which are found in adult bone marrow and newborn umbilical cord blood. Cord blood is the most enriched source of these cells. Even that source, however, is not enough for one adult bone marrow transplant because the total volume in cord blood is small. One of Bhatia's major goals is to discover which signaling factors can help researchers grow blood-forming stem cells in culture for therapeutic use.

Bhatia and his group knew from developmental biologists that blood cells develop out of an embryonic germ layer called the mesoderm. They hypothesized, says Krysta Levac, a research associate in Bhatia's laboratory, that the signaling factors involved in directing mesoderm cells to become blood tissue might help human blood-forming cells grow in a petri dish.

It turns out they do. Sonic Hedgehog, a protein ligand in the Hedgehog pathway; Bone Morphogenetic Protein-4, a ligand of the BMP pathway; and at least two ligands in the Notch pathway, Jagged-1 and Delta-1, help blood-forming stem cells survive and proliferate in culture. Bhatia and coworkers found that a member of the Wnt pathway, Wnt-5A, although ineffective in culture, enhances the ability of human blood stem cells to repopulate mouse blood in vivo.

Such results highlight the intertwining of multiple pathways in regulating stem cell fate. In the past, Levac says, "we've looked at pathways one at a time--in isolation. But we are now working on looking at them in combination." If BMP-4 increases the lifetime of stem cells and Jagged-1 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. "A lot of these pathways regulate really fundamental processes," says Lee L. Rubin, chief scientific officer at Curis, a biotechnology company cofounded by Harvard University stem cell researcher Douglas A. Melton. For example, the Hedgehog pathway "controls the balance between proliferation and differentiation. Basically it regulates how many cells you have and what kinds they are." That's why the three ligands of the Hedgehog pathway "are involved in pretty much the development of every tissue," Rubin says.

Curis was founded to explore how studying embryonic development in stem cells could lead to new disease therapies. Curis has specifically focused on identifying and developing drug candidates that either inhibit or activate the Hedgehog pathway.

Curis has some promising leads. A few activators of the Hedgehog pathway have shown potential in treating neurodegenerative disease, and Curis has partnered with Wyeth Pharmaceuticals to develop them. In addition, some inhibitors of the Hedgehog pathway may effectively treat basal cell carcinoma and solid tumors. Curis is working with Genentech on this project.

Rubin says that influencing a fundamental pathway like Hedgehog, which coordinates the activity of many other pathways, leads the body to respond in multiple ways. The complexity of the response can be daunting, but it also means Curis' drugs have the potential to effect multiple therapeutic responses. For example, Curis' Hedgehog inhibitors both directly block tumor cell proliferation and cut off the blood supply to tumors by inhibiting the production of growth and angiogenic factors.

Using stem cells to look for cancer therapies, as Curis has done, is logical because the same developmental pathways that work correctly in stem cells often work incorrectly in cancer cells. One chemical engineer has taken advantage of the similarities between cancer cells and stem cells by applying a novel biochemical tool developed for cancer cells to stem cell research.

Stem cell cultures, like cancer cell cultures, are rarely pure, says Julie Audet, an assistant professor at the Institute of Biomaterials & Biomedical Engineering at the University of Toronto. Cancer cell cultures constantly accumulate mutations, and stem cells constantly tend toward differentiation.

Performing a traditional biochemical assay requires a large population of (ideally) identical cells. When a heterogeneous population is used, measurements on all the cells--pure stem cells and differentiated cells--are averaged. That means "you don't have good information about what is going on in the stem cells," Audet says.

For her postdoctoral research, Audet looked into ways of measuring what is happening in a single cell. She found an assay at the University of California, Irvine, in the laboratory of cancer researcher Nancy L. Allbritton. The assay consists of first loading a cell with enzyme reporters--fluorescent peptides designed to be modified by only one cellular enzyme each. Once inside, the reporters are modified by their respective enzymes, the cell is lysed, and the contents of the cell are then separated and quantified by capillary electrophoresis.

The results indicate the level of activity of each targeted enzyme in just one cell, instead of averaging the characteristics of a heterogeneous population. Another "advantage of this approach is that you can monitor the activity of several enzymes at the same time," Audet says. At the moment, she is developing reporters for each enzyme in the Notch and Wnt signaling pathways.

THE LACK OF pure stem cell populations for research is not the only challenge of working with stem cells. Basic culture and maintenance of stem cell lines is demanding and tedious. Another team of chemical engineers is working on just this problem.

Human embryonic stem cells "grow very slowly, and our ability to preserve them is poor," says Sean P. Palecek, a chemical engineer at the University of Wisconsin, Madison. Few cells survive the suspension, freezing, thawing, and replating that occur during cryopreservation, the method used to preserve almost all types of cells. "Approximately 0.1% of the cells will survive these processes," Palecek says. In addition, the stress of the 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 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 (DMSO). Combinations of these stresses cause many cells to undergo cell suicide--or apoptosis--during cryopreservation.

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 of human 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 if the cells or their progeny are ever going to be transplanted into humans for therapeutic purposes.

Creating a synthetic matrix on which to grow human embryonic stem cells is just one of the chemistry-related challenges that face the stem cell research community. Finding the factors that such a matrix requires will take the collected effort of many types of researchers, from chemical 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."