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 cells. 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).