Year 2

The goal of the project is to find new ways to make adult cells such as skin cells into stem cells that have the capacity to differentiate into many tissues. The original methods to do this “reprogramming” used viruses, which can make the cells cancer-prone. We have developed new methods that do not use viruses and are expected to be safer. To initiate the studies, we constructed a new plasmid, or circle of DNA, that carries four genes that can stimulate adult cells to turn into stem cells. We also included a section of DNA that carries an “insert me” signal that causes the plasmid to become integrated into the cell’s chromosomes when an integrase enzyme is present. In order to introduce the plasmid DNA into cells, we employ a method that uses an electric shock to disrupt the cell membrane. This method, called electroporation, was used to introduce the reprogramming plasmid and a plasmid carrying the gene for integrase into human and mouse cells. After culturing the cells for one to three weeks, we observed colonies of cells that had the characteristics of embryonic stem cells. We proved that the reprogrammed cells were stem cells by performing many assays. For example, we showed that the reprogrammed cells expressed genes that were characteristic of embryonic stem cells and were different from the genes expressed in the starting cells. We also characterized the reprogrammed cells that we produced in terms of their ability to differentiate into different tissue types both in culture and when injected into mice. In a very stringent test of reprogramming, we demonstrated that the reprogrammed mouse cells were able to form many parts of the mouse body when injected into early embryos. These tests showed that we had produced high quality, reprogrammed stem cells by our new method. The finished method contains three steps and is called the triple recombinase strategy, because it uses three different recombination enzymes to achieve different steps. We use an integrase enzyme to add the reprogramming genes at certain preferred locations in the genome. After reprogramming is accomplished, we use a second enzyme to cut out the reprogramming genes precisely, since they are no longer needed or wanted. We then use a different integrase enzyme to insert a therapeutic gene into the preferred location formerly occupied by the reprogramming genes. This places the therapeutic gene in a position in the genome that will be expressed strongly. We are now refining our method to make it even easier, safer, and more efficient for producing reprogrammed human stem cells. We have also begun experiments to use the reprogrammed cells made with these methods to cure genetic diseases. For example, we are differentiating the reprogrammed stem cells into muscle precursor cells that could be used to form healthy muscle fibers in patients with limb girdle muscular dystrophy.