Compiled by BioPhotonics staff
LA JOLLA, Calif. – Before stem-cell-based regenerative medicine can advance,
scientists must understand how stem cells work and what kinds of cells they produce.
Salk Institute research recently uncovered a way to study neural stem cells by tagging
them with genetically incorporated “unnatural” amino acids, such as
those that emit green fluorescence. The neural stem cells differentiated into brain
neurons with the incandescent tag intact, allowing scientists to follow the process.
Although stem cells hold great promise for disease treatment,
it is difficult to study how they renew themselves and produce all of the body’s
cells, said Dr. Lei Wang, assistant professor and Frederick B. Rentschler Developmental
Chair in the institute’s Chemical Biology and Proteomics Laboratory.
“The ability to genetically incorporate unnatural amino
acids in stem cell proteins will accelerate our understanding of the signaling networks
that control these stem cells,” Wang said.
Although they had been used in bacteria in 2001 and in mammalian
cells in 2007, this marks the first time that unnatural amino acids (Uaas) have
been used in stem cells. The first step of the study was to see whether Uaas could
be incorporated into neural stem cells without disrupting their process of differentiation
– and, if that were possible, whether the fluorescent tag the team had inserted
would be carried into neuronal cells created by the stem cells.
Because added genes are often lost before a stem cell has a chance
to finish differentiation, current methodology for Uaa incorporation is not appropriate
for stem cells. To address this issue, the scientists developed a lentiviral-based
gene delivery method to incorporate the Uaas into proteins expressed in neural stem
They used the virus to deliver different components needed in
the Uaas technology, such as synthetic transfer RNA and enzymatic synthetase. They
could then load it with the third engineered molecule: an unnatural amino acid.
Chemically distinct from the 20 naturally occurring amino acids in the body, these
unnatural amino acids can be engineered for various desirable properties, such as
the ability to fluoresce.
Uaas were successfully incorporated into neural stem cells, and
the incorporation lasted through differentiation; these cells could produce neurons
carrying the fluorescent amino acid. Further studies demonstrated that these Uaas
could be used to help solve a biological question: How do voltage-sensitive ion
channels work in neurons?
“We are trying to understand how the electric field of cell
membranes can turn on or turn off protein activities – like a switch in a
house turns on or off lights,” Wang said.
To find the answer, the scientists embedded a fluorescent Uaa
into a domain that ion channels and other proteins use to sense the electric field
in neural stem cells. This produced neurons with the same embedded Uaa, allowing
the scientists to detect real-time changes in fluorescence intensity due to changes
in electrical current across the neuron.
The experiment, designed to demonstrate the power of Uaas in brain
cells, could be adapted for studying membrane proteins in other cells, no matter
where they exist in the body, Wang concluded. The new technique was detailed in
the June 16 online issue of Stem Cells (doi: 10.1002/stem.679).