How to ID human pluripotency

Stem cell researchers must take more care in identifying true pluripotency in reprogrammed human cells, according to a study published online today (October 11) in Nature Biotechnology. The paper outlines strict molecular criteria for recognizing pluripotency, and warns that relying on just a single marker will muddle the field.

Human embryonic stem cells
Image: Wikimedia commons,
Nissim Benvenisty

"All too often people in the human [stem cell] field use the most minimal criteria to call cells pluripotent," said linkurl:George Daley,;http://daley.med.harvard.edu/ a stem cell biologist at the Harvard Stem Cell Institute and Children's Hospital Boston who coauthored the study. "[The] colonies, on the surface, look like they're reprogrammed, but by stringent criteria are not." Scientists are getting better at reprogramming human cells into stem cells with embryonic-like properties -- known as induced pluripotent stem (iPS) cells -- but identifying those cells that have successfully reached true pluripotency may not be so easy. Researchers who work with mouse cells use molecular reporters integrated into pluripotency genes -- such as Fbx15, Oct4, or Nanog -- but there are currently no reliable parallels for human reprogramming studies. Simply looking at the cells is certainly not enough, and even common molecular markers of pluripotency can be misleading, Daley said. Using live cell imaging, Daley and his colleagues tracked various markers of pluripotency through the reprogramming process, and characterized the resulting cell types based on morphology and molecular makeup. Of the tens of thousands of human fibroblast cells in each well at the start of a reprogramming run, the majority of the cells formed colonies that were morphologically indistinguishable from embryonic stem (ES) cells, but only a minority of those cells was truly pluripotent, a series of in vitro and in vivo tests revealed. Based on the imaging results, the team divided the ES-resembling colonies into three distinct types -- dubbed type I, II, and III. The researchers injected each cell type into immunodeficient mice to perform a teratoma assay -- one of the "gold standards" of pluripotency in human stem cells, Daley said. They found that type III and most type II cells formed tumors that differentiated into multiple germ-cell layers, albeit to varying degrees -- an indication that they had indeed achieved some level of pluripotency. Several epigenetic factors, however, including hypomethylation on the gene promoters of NANOG and OCT4 and histone modifications characteristic of a pluripotent state, were only found in type III cells. "Our paper is a warning that if you want to be sure you are dealing with pristine iPS cells you need to do a host of things: teratomas, epigenetics, gene expression," coauthor and stem cell biologist linkurl:Thorsten Schlaeger;http://www.childrenshospital.org/cfapps/research/data_admin/Site799/mainpageS799P10.html of the Children's Hospital Boston wrote in an email to The Scientist. "That's cumbersome, but necessary to avoid problems, confusion, and conflicting results down the road." "The field is more eager to find the different methods to produce a higher yield of iPS cells," said molecular biologist Xiangru Xu of linkurl:Yale University,;http://www.yale.edu/ who was not involved in the research. "This study is emphasizing the quality rather than the quantity of iPS cells. It will eventually lead the field to produce better quality iPS cells, [which] will be very helpful to produce patient-specific cells." Despite pointing out the difficulties associated with identifying true iPS cells, the study does provide some tools for doing so, said cell biologist Mahendra Rao of linkurl:Life Technologies;http://www.lifetechnologies.com/ in California, who was not involved in the work. It describes in detail the molecular changes that cells undergo during reprogramming and, in doing so, provides a fairly reliable way to distinguish cells that are on their way to becoming truly reprogrammed. "It's a step in the right direction," Rao said. Furthermore, he added, because they are using live cell imaging, "you don't kill the colony to detect if it's truly reprogrammed, which means you can do it early in the process and select cells" that are likely to reach true pluripotency. Based purely on the molecular signature of the different cell types, the researchers were able to identify future iPS cells within just a week or two, which is much sooner (and easier) than conducting the teratoma assays. It is also "vastly superior" to another widely-used assay known as alkaline phosphatase staining, which this study shows can falsely identify cells as pluripotent, Schlaeger said. "I wouldn't trust any single marker," Daley said, "[but] this is a step towards identifying a molecular signature for pluripotency that we can be confident in. One day we'd like to be able to [provide a trustworthy] molecular surrogate" for true pluripotency.
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