'Unnatural' Chemical Allows Salk Researchers To Watch Protein Action İn Brain Cells

Researchers 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. They say this new technique, described in the June 16 online issue of 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.

"Stem 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, Dr. Lei Wang, assistant professor and Frederick B. Rentschler Developmental Chair in the Chemical Biology and Proteomics Laboratory.

"The 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.

The study's first author, Dr. 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 tube." These studies can also be conducted in real-time, says Shen, who is a postdoctoral researcher in Wang's lab.

Use 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.

Salk chemists, molecular and cell biologists, and experts in protein structure collaborated on this study, which was conducted in two stages.

The 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.

"Current 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. To solve that problem, the researchers developed a lentiviral-based gene delivery method to incorporate the Uaas into proteins expressed in neural stem cells. "The lentiviral gene therapy technique, which was pioneered by Dr. Inder Verma at Salk, can afford long-lasting expression through stem cell differentiation," Wang says.