Biologists from the Scripps Research Institute and the Genomics Institute of the Novartis Research Foundation have developed a high-throughput cell-based chemical screen to identify a small molecule that can maintain the self-renewal of mouse embryonic stem cells in the absence of feeder cells or other exogenous factors.
The finding is important because embryonic stem cells have recently become an attractive tool for high-throughput and high-content cell-based drug screening, as well as for basic disease and developmental biology research.
In addition, the cell-based screen and any resulting small molecules that promote embryonic stem cell self-renewal may pique the interest of companies that supply stem cells for research purposes, as evidenced by Invitrogen’s recent licensure of similar technology from Geron.
The potential for using embryonic stem cells as tools for basic cell biology research and drug discovery is well-established. However, the cells are daunting to use because of the time and money involved propagating them. This usually involves adding special serum mixtures and plating out so-called feeder cell layers in which the stem cells can grow.
As described in the Nov. 6 online edition of the Proceedings of the National Academy of Sciences, the Scripps and GNF researchers, along with scientists from Max Planck Institute for Molecular Biomedicine in Germany, screened 50,000 chemical compounds against a reporter murine embryonic stem cell line.
The cell line, derived from transgenic mice, expressed a GFP reporter molecule, but the cells are known to lose both their GFP expression and typical “compact-colony” morphology in the absence of feeder cells and another specific growth factor, leukemia inhibitory factor (LIF), the researchers wrote.
The scientists plated these cells in 384-well plates for one day with LIF, but then removed this growth factor and added compounds from a library of 50,000 heterocycle compounds. After six days of incubation, they analyzed the cells for GFP expression and morphology using a combination of FACS analysis, immunocytometry, histocytochemistry, and RT-PCR.
From the primary screen, they identified 28 compounds that maintained colony morphology and GFP expression, while 17 of these 28 also maintained the expression of multiple murine embryonic stem cell markers, the researchers wrote.
From these 17 compounds, they identified a class that maintained the phenotype of the cells in a dose-dependent manner, and eventually identified a previously uncharacterized heterocycle called SC1 that had 10-fold higher activity than other molecules with relatively low cellular toxicity.
The researchers further characterized the mechanism of action of the SC1 target and found that it likely exerts its effects by inhibiting the ERK1- and RasGAP-dependent signaling pathways.
“The key [factor] to consider is that mouse ES cells have been around for over 25 years, and on a day-to-day basis in the lab we still culture them using bovine serum, which is completely undefined,” said Alex Meissner, a postdoc studying embryonic stem cell differentiation at the Whitehead Institute for Biomedical Research who was not involved with the research but had seen the paper.
“You’re basically growing cells in something that has some key molecule in it that we know is required, but in general there is a lot of variation because you have all kinds of things in there,” Meissner added. “For most general mouse experiments, that works fine, because … you don’t necessarily need to avoid these other factors.
“But ultimately, if you want to grow human ES cells … finding these perfect combinations where you can keep these cells without any supporting feeders or anything you don’t know about is an important step forward.”
In addition, relatively little is known about the intracellular workings of stem cell self-renewal, and the research may demonstrate a way to help elucidate some of these pathways.
“Although significant progress has been made in recent years, we are still far from a complete picture of the dynamic regulatory circuitry that controls the self-renewal of ES stem cells,” the researchers wrote in the PNAS paper. “Consequently, unbiased cellular screens for small molecules or genes that regulate the self-renewal of ES cells may provide new insights into these processes and also facilitate practical applications of ES cells in research and therapy.”
“Ultimately, if you want to grow human ES cells … finding these perfect combinations where you can keep these cells without any supporting feeders … is an important step forward.”
There also may be big money in identifying feeder-free methods for stem cell renewal, as suppliers of embryonic stem cells are clamoring for such methods to offer their customers reliable, cost-effective ways to grow large quantities of human and mouse embryonic stem cells for research purposes.
A recent example of this is a decision in March by Invitrogen to license an intellectual property portfolio from Geron related to techniques for growing hESCs in the absence of feeder cells, and to specific media formulations that can be used for such feeder-free growth.
In the end, Whitehead’s Meissner said, it is important to find ways to propagate cells in the absence of feeders, but it may be even more crucial to discover methods for further differentiating cells under such conditions. The Scripps and GNF scientists were only able to show they could maintain self-renewal of the cells for approximately 10 passages.
“One key thing for human cells is of course you need to derive ESC lines,” Meissner said. “Not only do you have to maintain them, but you have to derive them in feeder-free conditions, or more defined conditions, which is much more difficult to do.”
It is unclear whether the Scripps and GNF researchers plan to extend their research to test whether they are able to further derive stem cells using the SC1 molecule. Multiple calls and e-mails to the paper’s co-authors were not returned. However, the scientists did write in the PNAS paper that they are currently exploring the effects of SC1 on human ES cells and adult stem cells.