by Monya Baker
Genetic vector is off in differentiated cells, on in stem cells
Most techniques to identify reprogramming human cells rely on researchers who know exactly what they are looking for: colonies that look like human embryonic stem cells. A new technique that causes reprogramming human cells to glow green may help open up the field to more scientists.
The most efficient gene transfer vectors are rapidly silenced in induced pluripotent stem cells, rendering many gene reporter constructs unusable. Researchers led by James Ellis of the Ontario Human iPS Cell Facility re-engineered these vectors to circumvent this problem so that they become active only as cells reprogram1. "We've managed to flip the specificity of expression," explains Ellis.
The lentiviral vectors Ellis used contain several elements: the promoter from a transposon called ETn, which is known to be highly transcribed in embryonic stem (ES) cells; binding motifs for known pluripotency genes Oct4 and Sox2; the gene for enhanced green fluorescent protein and a gene that allows cells to withstand doses of the antibiotic puromycin.
The researchers did a lot of testing to find exactly the best combination of various promoters and binding cassettes. The vector was active in ES cells but not in differentiated cells — when ES cells differentiated, the vectors became silent.
Next the researchers showed that this vector made finding reprogrammed cells easier. The number of colonies expressing the ES cell markers that could be expanded into cell lines increased eightfold to ninefold with mouse and human fibroblasts. Reprogrammed cells passed several tests for pluripotency, including teratoma formation. To show that the results were reproducible, the researchers reprogrammed cells from patients with Rett syndrome, an X-linked neurodevelopmental disorder. The generated cells could form cells representing several classes of tissue and could also become neurons.
Ellis believes this new vector could make the induced pluripotent stem cell field more accessible to scientists with little experience working with human ES cells. "In our system, they can look for the green colonies and use the puromycin cassette to get rid of the bad colonies," he says. Furthermore, treating the cells with puromycin in culture could kill off any cells that begin differentiating.
And the technology could also be used to evaluate both reprogramming and differentiation protocols, says Ellis. Plates of cells used in high-throughput imaging systems could help compare conditions. To find better reprogramming technologies, for instance, researchers would look for methods that turn more fibroblasts green. For differentiation technologies, they'd start with green, pluripotent cells and monitor how enhanced green fluorescent protein was turned off.
Better differentiation techniques are particularly important because all major applications of reprogrammed cells — drug screening, disease modelling and cell therapies — require differentiated cells. Ellis says the vector could even be modified to kill cells rather than turn them green. Not only might this create more homogenous cell populations, but also it could potentially be used to kill teratoma-producing stem cells in experimental therapies if something goes wrong.
Source: Nature