12 June 2003. In last week's PNAS online, researchers reported that murine embryonic stem cells exposed to pyrrolpyrimidines differentiate into neurons. This result raises hope that the fate of stem cells in vivo may one day be controlled by the use of small molecules.
Embryonic stem cells can be coaxed to differentiate into a variety of cell types, such as muscle, glia, and neurons. Traditionally, this has required that the cells be exposed to specific proteinaceous growth factors or cytokines (see ARF related news story), which are difficult to administer in vivo. While the small, cell-permeable retinoic acid has also been used to provoke differentiation, this pleiotropic compound causes the cells to differentiate into many different cell types.
Principal author Peter Schultz and colleagues from The Scripps Research Institute, La Jolla, and the Novartis Research Foundation, San Diego, California, set about to identify small molecules that could lead stem cells specifically down the path to full-fledged neurons. First author Sheng Ding and coworkers used genetic engineering to equip mouse stem cells with a luciferase reporter gene that is under the control of the neuronal tubulin gene promoter. Because this promoter is only active in neurons, the authors could use luciferase activity to identify neuronal phenotypes among the stem cells and their progeny. Using high-throughput screens to expose these modified stem cells to thousands of small molecules, Ding and colleagues found chemicals that promoted neuronal differentiation.
From this preliminary analysis, the authors chose to focus on pyrrolpyrimidines. By tweaking the chemical substituents attached to the pyrrolpyrimidine backbone, the authors found a compound, dubbed TWS119 that could induce neuronal differentiation in 40 to 60 percent of the stem cell population. In these cells, luciferase activity was accompanied by positive staining for a variety of neuronal cell markers, including TuJ1, NeuN, glutamate, and neurofilament-M. The authors also report that TuJ1-negative cells tested negative for muscle and glial cell markers, but did test positive for nestin, a sign that the cells are neuronal progenitors.
Curious to know how TWS119 works, Ding and colleagues used an affinity matrix to isolate stem cell proteins that bind to the compound. Only two proteins bound with high affinity, and the authors used mass spectroscopy to show that these are both isoforms of glycogen synthase kinase-3β (GSK3β). This kinase is known to be involved in the "wingless" signaling pathway, phosphorylating the pathway component β-catenin and stimulating its degradation. When Ding and colleagues examined TWS119-treated pluripotent cells, they found that levels of β-catenin had increased over 10-fold, consistent with inhibition of GSK3β. β-catenin is already linked to neurogenesis (see Walsh section in ARF related news story) and to presenilin (see de Strooper section in ARF related news story). It also is worth noting that GSK3β has been implicated in the phosphorylation of the protein tau, which is found in the neurofibrillary tangles that often occur with Alzheimer's disease (see ARF related news story).
“This is a very interesting idea to direct cell lineage,” commented Kiminobu Sugaya, University of Chicago, “though I read this paper with a little bit of surprise because regulation of only one enzyme, GSK3β, seems sufficient to lead embryonic stem cells to neurons. I would like to see further investigation of the mechanism of action of this small compound” he added.—Tom Fagan.
Ding S, Wu TYH, Brinker A, Peters EC, Hur W, Gray NS, Schyltz PG. Synthetic small molecules that control stem cell fate. Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7632-7. Abstract