Live-cell imaging techniques move toward a definitive assay for pluripotency
The next tools for reprogramming cells to an embryonic-like state might just be a camera and a set of fluorescently tagged antibodies, according to a study reported in Nature Biotechnology.
Researchers literally took pictures of more than a million human cells as they transitioned from the cultured skin cells known as fibroblasts into colonies of induced pluripotent stem cells. As expected, many similar-looking colonies appeared, but only a very few consisted of fully reprogrammed iPS cells. After tediously assessing which were which, researchers led by Thorsten Schlaeger and George Daley of the Harvard Stem Cell Institute in Massachusetts went back and analyzed the images to figure out how to predict which colonies would produce high-quality iPS cell lines.
Robert Blelloch, who studies reprogramming at the University of California San Francisco, sees immediate practical applications. “It means that you can focus down on the most promising colonies and not assay everything.” Currently, he says, many evaluations of techniques to boost reprogramming rates lump some partially reprogrammed cells together with fully reprogrammed ones. With better markers of pluripotency, he says, “you can look at the dish and count” and be more confident of your results.
The analysis shows that individual markers alkaline phosphatase, SSEA-4, GDF-3, hTERT and NANOG sometimes used to assess reprogramming rates can be misleading. In other words, colonies that appear to be reprogrammed may actually not be, says Schlaeger. “There have been a lot of bad studies in the stem cell field, and our study highlights the need for rigorous analysis.” Instead, Schlaeger and colleagues identify a series of markers that can place cells from morphologically similar colonies into three types; two could form teratomas in immunocompromised mice, a standard assay for testing iPS cells, but only one of these shows epigenetic modifications indicating bona fide iPS cells.
“I think these [kinds of] molecular criteria are going to be the best way to characterize iPS cells,” says James Ellis, who directs the Ontario Human Induced Pluripotent Stem Cell Facility in Toronto. Lead author Schlaeger isn't so sure. “It's too early to tell if this could replace the teratoma assay,” he says. For now, he says, an assessment of how cells will behave is more informative than a collection of markers.
However, Schlaeger does think that more researchers can use live imaging and antibodies to find more markers in reprogramming human iPS cells. When studying reprogramming in mouse cells, scientists routinely link the expression of important genes to the production of fluorescing proteins, which allows them to see when a gene is active. This technique is much more difficult in human cells, so the Harvard researchers used fluorescently-tagged antibodies instead. These could be added directly to growing cells to reveal the presence of particular molecules on cell surfaces. An automated microscope scanned and recorded images as the cells grew. “It's surprisingly easy,” Schlaeger says. “It worked the first time we did it.”
However, antibodies and live cells weren't by themselves sufficient to pinpoint truly reprogrammed colonies. Standard techniques for inducing cells to pluripotency rely on viruses to insert copies of four reprogramming genes into cells, and the researchers tied expression of each of these genes to expression of green fluorescent protein. As cells reprogram, they turn on their own pluripotency machinery and silence the introduced genes; the subsequent dimming of GFP is an important marker of reprogramming. The live-image technique can't be used to evaluate newly reported techniques that don't require viruses. But the expression of some genes may be able to stand in for viral silencing, Schlaeger says, and live-cell imaging can be combined with destructive sampling techniques that can detect them.
However, the live-imaging approach may be of even more use in studying the beginning of reprogramming rather than the end product, says Schlaeger. “It allows us to identify earlier stages, so we can look at molecular events in these very rare cells.” The technique allows researchers to watch just-emerging cell clusters and watch as as markers turn on and off. “It's extremely important to follow the fate of human iPS cells as they reprogram,” says Ontario's Ellis. “This is really the first paper to do that on single human iPS cell colonies induced using the standard retrovirus vector reprogramming approach.”
Chan, E.M. et al. Live cell imaging distinguishes bona fide human iPScells from partially reprogrammed. Nature Biotechnol. 11 October 2009; 10.1038/nbt.1580
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Baker, M. Finding real iPS cells early. Nat Rep Stem Cells (2009). https://doi.org/10.1038/stemcells.2009.131