New rapid technique purifies human embryonic stem cells using FRET
University of California, San Francisco (UCSF) scientists introduced into a stem cell culture a pair of DNA snippets containing part of a gene, called Oct4, which is essential to all embryonic stem cells. The gene also allows cells to form tumors called teratomas, but it is not active once a stem cell starts down the road to becoming a specialized cell type. At one end of each DNA snippet, or "molecular beacon," the researchers attached a fluorescent protein, and, at the other end, they engineered the DNA so that it could "quench" the fluorescent light using a process known as fluorescence-resonance energy transfer (FRET).
When introduced into the cell culture, the two DNA fragments naturally sought to bind, or anneal, to similar genetic sequences in the messenger RNA, or mRNA, made by the cell's Oct4 gene. However, they could do so only if the gene was turned on, as it is in teratoma-forming cells—not in stem cells already destined to become specialized tissue. When the two DNA snippets annealed to an Oct4 mRNA, their fluorescent proteins lit up, and a cell-sorting machine then fished out the fluorescently tagged cells—but no others. They reported separating out the desired stem cells from the teratoma-forming cells at a rate of about 25,000 cells per second.
The research finding is currently published online in the journal Stem Cells and Development, and will appear later this year in a print edition of the journal. Embryonic stem cells, which replicate indefinitely in the culture dish, are capable of forming almost any tissue in the body. Over time, they begin to specialize as specific cell types, such as cardiomyocytes of the heart or neurons of the brain. One goal for stem cell therapy is to be able to identify cells that have begun to specialize in a particular way so that they could serve as a source of cells to repair specific damaged tissues.
The researchers say they expect that the same approach could be used to separate and purify different types of cells as they advance from the stem cell state into neurons, heart cells or any other type of tissue needed for future stem cell therapy.
Lead author on the paper is Frank W. King, PhD, who was a post-doctoral fellow with Bernstein at the UCSF Cardiovascular Research Institute (CVRI) when this research was done. Co-authors on the paper are Walter Liszewski, BA, and Carissa Ritner, BS, both staff research associates in the Bernstein Lab at the CVRI.
The research was supported in part by the state-funded California Institute for Regenerative Medicine (CIRM). The technique has been disclosed to the UCSF Office of Technology Management for potential licensing, as well as to CIRM. In addition to funding from CIRM, the research was supported by the National Heart, Lung and Blood Institutes.