Researchers have developed a new imaging technology to study stem cells as they proliferate and differentiate, according to a recent paper in Circulation Research (1). The technology deals with limitations of previous cellular imaging techniques and paves the way for imaging cell lines that could be used safely in stem cell therapies, the authors say.
Both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) can differentiate into any somatic cell type, and they have shown promise in cell replacement therapies. But "it is vital that we be able to track these cells both in real time and over time" in order to better understand their behavior and potential, said study author Joseph Wu of Stanford School of Medicine in Palo Alto, CA.
To visualize cellular behavior, researchers insert reporter genes, such as those for fluorescent proteins, into the genomes of the cells. These reporter genes typically insert randomly into the genome.
Random integration of reporter genes is problematic for a few reasons. Some cells have single copies of the reporter gene while others will have multiple copies. And, depending on where the reporter is inserted, expression can be unstable, and can even damage the cells.
Such issues "not only are severely detrimental to the health of the cell, but also add confounding factors to the study of the modified cells," Wu said.
To get around this, Wu and his co-workers used a technology called human genome editing to insert reporter genes into hESC and iPSC genomes. In genome editing, an engineered zinc finger nuclease creates a double-stranded break in the target DNA, and the reporter gene is then integrated at the break.
The researchers selected an insertion site in a gene on chromosome 19 called PPP1R12C, which had been proven inessential to the cells' behavior and unlikely to affect transcription of other genes. They inserted three reporter genes into this locus – for fluorescence, bioluminescence, and positron emission tomography (PET) imaging.
Wu and his colleagues found that the edited stem cells remained pluripotent and maintained reporter gene expression for up to two months. They imaged the cells successfully bothin vitro and in vivo. They also differentiated the cells into cardiomyocytes and epithelial cells – two cell types that have shown promise in treating myocardial infarction – and found they were indistinguishable from unmodified cells, suggesting they could potentially be used clinically.
Editing stem cells is not without its technical challenges, however, said Wu. Cell lines need to be grown from a single edited cell, which can be difficult, and the entire process takes a month or two. (Random integration of a reporter gene takes as little as a day.) But because of the limitations of random integration, being able to insert reporters via genome editing is "a very necessary tool," he added.