University of California, San Francisco (UCSF) researchers have developed a high-throughput sorting method to remove pluripotent cells, which can lead to the formation of teratomas from stem cell cultures after transplantation. The new method adapts a dual fluorescence resonance energy transfer (FRET) reporter system for a high-throughput cell sorter, avoiding the alteration of the cells’ genome by inserting a reporter gene.Stem cell sorting has traditionally relied on a reporter cassette inserted into the stem cell genome by lentiviral infection. The cassette uses the promoter of the gene of interest to activate a fluorescent reporter gene like GFP. “That’s an effective way to identify those cells, but you’ve changed the stem cell genome,” said Harold Bernstein, professor of pediatrics at UCSF and senior author of the paper. “You’ve introduced an alien piece of DNA.” Reporter genes that are inserted into a cell’s genome have the potential to corrupt that cell’s behavior. “There’s a real reluctance to use genetically engineered cells like that in any kind of clinical application,” said Bernstein.
To avoid corrupting the genome, the Bernstein laboratory developed a new method to identify undifferentiated cells using FRET molecular beacon technology. Each beacon was designed to fluoresce when annealed to a particular target sequence. In the group’s first experiment to identify undifferentiated cells, the target sequence was oct4, one of the well-expressed genes associated with pluripotency. Only when both of these beacons anneal to oct4 is a FRET signal produced when one dye excites the other.
Although used to monitor gene expression in microscopy, FRET had not been applied to high-throughput fluorescence-actived cell sorting (FACS )methods. Fluorescence-activated cell sorters—which process over 25,000 cells per second—have less time to capture the fluorescent signal than microscope counterparts. “It took a lot of experimentation to come up with exactly which fluorescent dyes could be used that gave a strong enough emission signal that could be captured by the cell sorter,” said Bernstein.
After the sorting, the group observed the cells to verify that the molecular beacons did not modify them in any way that would affect cell behavior. Over time, the researchers could no longer detect the beacons and, since the beacons did not divide with the cells, they were diluted out of the culture. The authors believe that the beacons were expelled from the cells. “They don’t hang around, so you have to worry about what else they’re going to do,” said Bernstein. “Mammalian cells will spit out episomal plasmid DNA if there’s not selective advantage to them keeping it. So we’re thinking a similar process probably happens here.”
The Bernstein laboratory is now working on adapting this method to identify and sort stem cells that are already committed to differentiating into specific tissue types. The lab is specifically interested in muscle tissues as the group’s research goal is to understand the regulation of cardiac and skeletal myogenesis and repair.
The new gene targets are proving to be a greater challenge because of their expression levels. Though oct4 is well expressed in embryonic stem cells, other new target genes in differentiated cells are expressed at much lower levels. This creates signal-to-noise ratio issues that the group will have to overcome. According to Bernstein, the solutions will come from further experimentation with the design of the molecular beacons and with the cell sorting equipment.
“We’re also looking for industry partners who are interested in developing next generation cell sorting to see if there’s some way that we can tweak the equipment to make it more sensitive,” said Bernstein.
The paper, “High throughput tracking of pluripotent human embryonic stem cells with dual fret molecular beacons,” was published online Jul. 12, 2010 at Stem Cells and Development.