Photo of neurons differentiated from neural stem cells HCN-A94 with an unnatural amino acid incorporated. Photo: Courtesy of Dr. Bin Shen of the Salk Institute for Biological Studies
at the Salk Institute have been able to genetically incorporate
“unnatural” amino acids, such as those emitting green fluorescence, into
neural stem cells, which then differentiate into brain neurons with the
incandescent “tag” intact.
say this new technique, described in Stem
Cells, may help scientists probe the mysteries of many different kinds
of stem cells in humans as well as the cells they produce. This could be
a boon to both basic and clinical research, such as helping to speed
development of stem cell-based regenerative medicine.
cells hold great potential for the treatment of various diseases, yet
it has been hard to study how they self renew and produce all of the
body’s cells,” says the study’s senior author, Lei Wang, assistant
professor and Frederick B. Rentschler Developmental Chair in the
Chemical Biology and Proteomics Laboratory.
ability to genetically incorporate unnatural amino acids in stem cell
proteins will accelerate our understanding the signaling networks that
control these stem cells,” he says. Thorough understanding of these
mechanisms is critical for safe and reliable stem cell therapeutics.
study’s first author, Bin Shen, adds that the incorporation of
unnatural amino acids “allows researchers to study a particular protein
in a living cell or organism, compared to the traditional biochemical
methods which are conducted through in vitro settings such as a test
These studies can also be conducted in real time, says Shen, who is a postdoctoral researcher in Wang’s lab.
of unnatural amino acids (Uaas) was developed by Wang and his
colleagues, and was first used in bacteria in 2001, and in mammalian
cells in 2007. This is the first report of its use in stem cells.
chemists, molecular, and cell biologists, and experts in protein
structure collaborated on this study, which was conducted in two stages.
first stage was to see if Uaas could be incorporated into neural stem
cells, without disrupting their process of differentiation, and if so,
would the fluorescent tag they inserted be carried into neuronal cells
created by the stem cells.
methods for Uaa incorporation are not appropriate for stem cells,
because the added genes are often lost before the stem cell has a chance
to finish differentiation,” Wang says.
solve that problem, the researchers developed a lentiviral-based gene
delivery method to incorporate the Uaas into proteins expressed in
neural stem cells.
lentiviral gene therapy technique, which was pioneered by Dr. Inder
Verma at Salk, can afford long-lasting expression through stem cell
differentiation,” Wang says.
virus was used to deliver different components needed in the Uaas
technology. These consisted of a synthetic transfer RNA (tRNA) that
cells use to incorporate amino acids into a protein that is being built
inside a cell. The second molecule is an enzymatic synthetase that can
recognize the engineered tRNA, and load it with the third engineered
molecule—an unnatural amino acid. These amino acids are chemically
distinct from the 20 amino acids that naturally exist in the body; they
can be engineered for different desirable properties, such as to
you have a stem cell line stably incorporating Uaas, you can custom the
Uaa to study stem cell biology,” Wang says. “You also get the bonus of
obtaining various mature cells with this ability through
differentiation, such as neurons, which are difficult to insert Uaas in
and expensive to procure in large amounts.”
the first set of experiments, the researchers found out that Uaas were
successfully incorporated into neural stem cells, the incorporation
lasted through the differentiation, and these cells then produced
neurons carrying the fluorescent amino acid.
second set of experiments was to demonstrate how these Uaas can be used
to help solve a biological question. The researchers wanted to know how
voltage-sensitive ion channels, which are pore forming proteins, work
in neurons. In these nerve cells, membrane ion channels respond to
changes in electric current—the charged signal that is passed between
neurons—that either activate or silence the neurons.
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 says.
study this phenomenon, the researchers embedded a fluorescent Uaa into a
protein domain that ion channel and other proteins used to sense the
electric field in neural stem cells, which produced neurons with the
same embedded Uaa. They could then watch, in real time, the fluorescent
tag given changes in electrical current across the neuron. “We detected
changes in fluorescence intensity of the Uaa when the neurons were
stimulated, and these changes are dependent on where the Uaa was
incorporated, which hint that different positions of the protein are
moving into or outside of the membrane in response to the electric
says this experiment, designed to demonstrate the power of Uaas in
brain cells, can also be adapted to study other membrane proteins in
other cells, no matter where they exist in the body.