27/04/10

BioTechniques

A group of scientists from the Scripps Research Institute have demonstrated that two drugs—thiazovivin (Tzv) and pyrintegrin (Ptn)—can keep human embryonic stem cell (hESC) cultures alive longer than traditional supplements. Without the addition of these two drugs, the activity of E-cadherin—a cell adhesion protein prevalent in the cells—is interrupted during passaging and the cultures die. The team, led by Sheng Ding, an associate professor of chemistry at Scripps, published their findings in the Proceedings of the National Academy of the Sciences (PNAS).

“This paper addresses a long-standing mystery,” said Ding in a press release. “Scientists have been puzzled by why human embryonic stem cells die at a critical step in the culture process.”

Pluripotent hESCs are notoriously difficult to grow in culture, and cultures often die after passaging—that is, after moving cells from one medium to another. “The current techniques to keep these cells alive are tedious and labor-intensive," said Ding. “Keeping the cells alive is so difficult that some people are discouraged from entering the field.”

Finding the right medicine

To help hESCs survive passaging, Ding’s team tested about 50,000 synthetic compounds using a high-throughput phenotypic screening method. This method uses fluorescent tags and high-resolution microscopy to identify target proteins produced by the cells in response to particular compounds. The researchers found that Tzv and Ptn individually helped the hESCs survive more than 40 passages.

Ding’s lab uses chemical tools, including small drug molecules, to study stem cell cultures. “Without such an approach, it’s not easy to look at this question,” Ding told BioTechniques. “To have a handle on the problem, we need to have the [right] tools to look at the problem. With chemical tools, we can dissect the mechanism.” He believes that his chemical biology approach gives his laboratory an advantage over other laboratories that only use biological tools such as immunostaining, which uses antibodies to target and vizualize proteins of interest. Instead, Ding’s team uses small molecules that interact with proteins and alter binding sites on enzymes. Through these molecular interactions, researchers can get a better look at underlying biological mechanisms.

However, finding which drugs would help hESC cultures was just a first—and minor—step, according to Ding. The real difficulty was understanding how these two molecules interact with the cultures to keep them from dying. “Those mechanistic studies are more challenging because cell biology is very complex,” said Ding. “That’s why for more than 10 years, nobody had any idea why the cultures die.”

Understanding how the drugs work

When Ding’s team treated dissociated cells in cultures with either drug, the cells attached to the culture plates within in a few hours. The researchers suspected that the rapid reattachment was the result of the drugs stimulating integrin receptors. Integrins communicate with growth factors to activate enzymatic pathways for self-renewal and provide information about the external environment to the cell. Based on that information, the cell attaches to another surface, moves, dies or differentiates. To test their theory, the team cultured cells with the drugs, and an integrin antibody and a drug-free control group. Analysis of the cultures showed higher levels of the antibody bound to integrin in the drug-exposed cultures than in the control.

While studying the drugs’ effect on integrin, the researchers noticed that cells treated with only Tzv stayed alive after being passaged on gelatin-coated plates by forming floating clusters. But cells treated with only Ptn died after being passaged onto gelatin-coated plates. Suspecting that Tzv might be targeting another protein in addition to integrin, the researchers looked at E-cadherin, a transmembrane protein that helps cells stick to each other and influences floating cluster formation. After passaging onto gelatin-coated plates, Ding’s team compared an hESC culture treated with only Tzv with a control group treated with an E-cadherin antibody and Tzv. They found that while the control culture died, the Tzv-treated culture did in fact contain higher concentrations of E-cadherin.

Improvements to hESC culturing techniques are just the first of many steps toward better stem cell treatments, according to Ding. “Stem cell biology has many hurdles to overcome before we can really use them for therapeutic applications,” he said. “We still need to have an improved and better method to differentiate these useful human embryonic stem cells into functional cell types that would be ultimately useful for treating patients.”

The paper “Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules” was published online ahead of print in the Apr. 20 edition of PNAS.