Stem Cells Methods For Growth And Maintenance
by Mike MayStem cells took the scientific world by storm, and created a stir in the political world as well. In July of this year, the U.S. Senate passed H.R. 810, a bill that would have allowed federally funded research on any line of human embryonic stem cells for therapeutic purposes. The bill was vetoed by the president, and it is not anticipated that there will be enough votes to override the veto. Thus, scientists who want federal research money must stick with lines developed before 9:00 p.m. Eastern time on August 9, 2001.

The scientific tempest continues to blow, however. The biggest challenge remains finding new ways to grow and maintain stem-cell lines. Currently, one stem cell cannot be efficiently turned into many, and stem cells in culture tend to differentiate if the conditions do not stay just right. As Peter Mountford of Stem Cell Sciences (Edinburgh, UK) says, “The single biggest obstacle to commercial and clinical applications of stem cells is the lack of a robust ex vivo cell-culture system.” From a clinical perspective, scientists face an added issue: getting all animal products out of stem cells. In effort to meet all of these goals, many groups are developing new techniques.
Figure 1. Human neural stem cell image (courtesy Stem Cell Sciences).

Going synthetic

Scientists must keep stem cells growing and undifferentiated. To do that, researchers generally follow a basic approach: put stem cells in a culture dish that has been coated with cells, usually mouse skin cells, and fill it with medium. Scientists call the cell coating a feeder layer, and it provides a surface for attachment and adds nutrients to the medium. The animal components in the medium and feeder layer, however, might infect or contaminate the stem cells in some way, such as passing along mouse viruses or other substances. The animal cells can also cause potential regulatory problems down the road. For example, if someone uses a stem-cell product developed with animal cells during any part of the process, the U.S. Food & Drug Administration requires follow-up for the life of the patient. That is not the case if the cells are entirely animal-free. In addition, growing stem cells in the presence of feeder cells or animal products, such as fetal-calf serum, can trigger persistent variation in stem-cell growth, because of batch-to-batch variation in the uncharacterized support components.

Consequently, scientists want a completely defined system one based on chemicals. In creating such a system, however, researchers must still avoid problems. Tenneille Ludwig of the University of Wisconsin and the WiCell Research Institute (Madison, WI) says, “We need to work on decreasing spontaneous differentiation in culture and improving the cloning efficiency.” That is, scientists want the cells to remain as stem cells and to make more of them. “Right now,” says Ludwig, “the differentiation rate is fairly low under the right conditions, but the cloning efficiency is less than one percent.”

In addition to regulatory hurdles, scientists want an animal-free system to reduce variability. Both animal-based medium and feeder cells create considerable variability in culturing stem cells. A variety of such systems exist. For example, Tom Okarma of Geron (Menlo Park, CA) points out that his company can grow stem cells using chemically defined, serum-free reagents without any feeder cells. This patented system keeps the cells growing and undifferentiated. “That was one of the hardest things to achieve in developing this technology,” says Okarma.

But how do you know if stem cells differentiate? To keep track, Geron collaborated with Celera (Rockville, Maryland) to develop a library of genes that get expressed in undifferentiated cells and ones that turn on when the cells differentiate. “That gives a complete genomic signature of what undifferentiated cells should look like,” says Okarma. In addition, Geron forged a deal with Corning (Corning, NY) to create a synthetic surface, based on laminin, a human-matrix protein. All of this work demands deep investments. So far, Geron has spent $100 million on developing their human embryonic stem-cell platform and products.

Figure 2. Human embryonic stem cells (hESCs) are pluripotent stem cells that can differentiate into any and all cell types and tissues in the body. The photomicrograph shows hESCs as well as three types of differentiated cells made from hESCs. Each differentiated cell type exemplifies one of the three tissue layers from which all cells in the body are derived. The hESCs express telomerase and can therefore replicate indefinitely in the undifferentiated state. This self-renewal capability is a unique characteristic that distinguishes hESCs from all other human stem cells discovered to date and allows them to be a potential source for the manufacture of all cells and tissues of the body. (Courtesy Geron Corp.)
Academic insights

Many companies turn to academic scientists for input. Mountford says, “Since 1994, Stem Cell Sciences has aligned itself with academic centers at the cutting edge of understanding the biology of stem cells.” He adds, “From them, we get leads of what might be the appropriate factors to add to culture medium for growing stem cells well, and to develop the simplest medium possible.” Right now, Stem Cell Sciences focuses on three types of cells: embryonic stem cells, neural stem cells, and stem cells derived from the fat tissue of young children, and each requires different culture environments. Other stem-cell scientists work on many other cell types, including cardiac, epithelial, hematopoietic, pancreatic, hepatic, and so on. Mountford and his colleagues start with amino acids, salt, vitamins, and other chemicals to create the basic medium. Then, depending on the cells, the scientists add growth factors and other components. “Quite often, getting the right combination takes quite a bit of tinkering,” says Mountford.

In Wisconsin, Ludwig and her colleagues developed a culturing method that uses a medium composed of materials from humans. Their system does not even need a feeder layer. Still, the cells do need a matrix to grow on, and the Wisconsin team developed one that is not animal-based. Nonetheless, Ludwig says, “It is absurdly expensive.” When they want to reduce cost, Ludwig and her colleagues still use a matrix created from animal proteins.

Ways to use Wnt

To keep stem cells in culture, they must renew themselves, but that proves tricky. Nonetheless, scientists might learn to control renewal with Wnt proteins. “These proteins bind receptors that turn on downstream genes that participate in many developmental processes,” says Tannishtha Reya of Duke University Medical Center (Durham, NC). “When we started looking at this, it wasn’t clear that Wnt played a role in hematopoietic stem cells.” But it does. “We now have in vivo and in vitro evidence suggesting that this growth factor functions to regulate renewal and growth,” says Reya.
Figure 3. 103 view of a colony of undifferentiated human embryonic stem cells being studied in developmental biologist James Thomson’s research lab. The embryonic stem cell colonies are the rounded, dense masses of cells. The flat, elongated cells in between the embryonic stem cell colonies are fibroblasts that are used as a “feeder layer” on which the embryonic stem cells are grown. (Courtesy University of Wisconsin-Madison.)

A better understanding of Wnt’s role could trigger new ways to control the renewal of stem cells. These cells renew easily in the body but not so easily in a dish. Reya already knows that shutting off the Wnt pathway reduces the lifespan of stem cells and leads to their exhaustion. In addition, if she activates those pathways, the stem cells grow for a longer period of time without differentiating. “We are trying to look at other signals too,” says Reya. “We would like to identify the factors that control stem-cell growth and regulate the choice between renewal and differentiation.” Eventually, those factors could be used to control what stem cells do and even the types of cells that they create. The Wnt system might even work for a variety of stem cells. “A lot of data suggest that Wnt seems to be used fairly generally for a lot of stem cells,” says Reya. But it may work in different ways with different cells, especially if placed in the context of other signals.

Advances from space

In some cases, the research on maintaining stem cells involves new technology, instead of new chemical concoctions. For example, Donnie Rudd of Regenetech (Sugar Land, TX) and his colleagues turned to NASA for a new approach. Rudd and his colleagues take peripheral blood, separate the stem cells with commercially available kits, and then put them in a bioreactor that is the size of a shoebox.

“The bioreactor is a rotating cylinder,” says Rudd, “like a soup can on its side.” Basically, it works in a way that is the opposite of how a centrifuge works, that is, instead of swinging cells to the side of the cylinder it keeps them in the middle, essentially suspended in medium. “It is a simulated weightless condition,” Rudd explains. “It simulates results found in MIR and on space-shuttle flights.”

The stem cells also experience a magnetic field that resembles that of outer space. “It causes the stem cells to expand,” says Rudd, “and more important, they do not differentiate.” Rudd and his colleagues license this technology to pharma and biotechs.

Spreading the sources

A key especially in the political battle involves the source of stem cells. “For human embryonic stem cells,” says Robert Lanza of Advanced Cell Technology (Alameda, CA), “the main obstacle today is that getting them requires the destruction of an embryo, and that is caught up in serious controversy.” To bypass that issue, Lanza and his colleagues developed a technique that does not destroy or even harm an embryo.

Physicians use so-called pre-implantation genetic diagnosis to check embryos created through in vitro fertilization for genetic defects before implanting. These tests use a blastomere, and Lanza’s team says the same cell can provide human embryonic stem cells. “We would propose taking that cell that is usually destroyed for the test,” says Lanza. “We’d let it grow overnight, and it would grow a stem-cell line that closely matches the child.”

Wherever the stem cells come from, though, scientists face political and public battles. “People think we can have therapeutic cures tomorrow,” says Ludwig, “but that is not the scientific reality.” It could be decades before stem cells turn into approved therapeutics. Nonetheless, these cells could be used much sooner in drug discovery and for learning more about the developmental processes behind good health and disease. So, finding new and better ways to grow and maintain stem cells is a valiant battle. It could create the foundation for remarkable therapies in years ahead and for new knowledge today.