Embryonic stem cells open up exciting new prospects for medicine, because they can differentiate into any tissue in the body. Therefore, they have the potential to be used to repair faulty tissues in diseases like diabetes, heart disease, and neural disorders. Furthermore, stem cells can be corrected by gene therapy and transplanted, in order to treat a wide variety of genetic diseases, such as sickle cell anemia. However, embryonic stem cell research has been difficult because of the technical and ethical problems involved in obtaining these cells from human embryos, as well as the need to transplant cells that are immune-matched to the recipient patient. The solution to these challenging biological and ethical problems may emerge from recent findings that show that cells that behave like embryonic stem cells can be derived from ordinary cells easily obtained from a patient, such as skin cells. This process is known as reprogramming. This breakthrough means that embryos may no longer be required to generate the stem cells needed for exciting new therapies. However, the methodology that is currently used to create reprogrammed cells involves introducing many viruses into patient cells. This procedure is itself dangerous and can lead to tumors and other abnormalities in the cells. As a result, the reprogrammed cells created to date are not suitable for use in the clinic. This proposal seeks to solve this problem by creating a novel method for introducing the reprogramming genes into one safe place in the chromosomes that will have no adverse effects on the cells. In these experiments, a simple, safe way to make reprogrammed cells without viruses will be developed. The reprogrammed cells made by this method will be thoroughly tested to ensure that they have all the beneficial properties of embryonic stem cells and are safe to use. The emphasis will be on generation of human reprogrammed cells that are safe and effective in therapies. Reprogrammed mouse cells will also be generated, for use in testing in mice before human clinical trials. The reprogrammed cells will be evaluated for their ability to differentiate in culture into tissues such as nerve, heart, and blood cells. The cells will then be tested for their capacity to cure a genetic disorder in a mouse model. Success in these experiments will provide a simple and safe method to generate reprogrammed stem cells and will speed the use of these cells in a wide variety of clinical applications.
Human pluripotent stem cells derived from ordinary adult cells are a scientific breakthrough that could speed medical advances to the public. These “reprogrammed” stem cells, made from ordinary cells, could remove the technical and ethical impasses that have delayed advances with stem cells that are derived from human embryos. However, current methods to make reprogrammed cells utilize viruses that are themselves dangerous. This proposal will apply new, California-invented technology, in the form of a novel gene addition system, to create an easy and safe method to make reprogrammed stem cells without the use of viruses. By replacing the current ~20 random viral integration sites with one safe, defined integration site, the resulting stem cells are likely to be suitable for clinical use, without the fear of tumors or other abnormalities. This project is feasible in all its elements, highly relevant to the goals of CIRM, and will result in a variety of new lines of pluripotent human stem cells. The availability of high-quality stem cells, made from ordinary patient tissue, will allow researchers to move more quickly to develop safe, effective, stem cell therapies for the people of California. This highly innovative project could create a leap forward for the entire stem cell field and greatly speed clinical applications. Therefore, this application is of great importance to California.
The goal of the project is to find a new way to make adult cells such as skin cells into stem cells that have the capacity to differentiate into many tissues. The original method to do this “reprogramming” used viruses, which can make the cells cancer-prone. We are developing a new method that does not use viruses and is expected to be safer. To initiate the studies, we constructed several new plasmids, or circles of DNA, that carry two to 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. These plasmids have the potential to reprogram cells, and we are testing them to see which ones work the best. The next objective was to find methods to introduce the plasmid DNA efficiently into the mouse and human cells that we wanted to reprogram. We tried a series of chemical agents and also tried a method that uses an electric shock to disrupt the cell membrane. The latter method, called electroporation, worked the best. Using these methods, we verified that the reprogramming genes that we placed on the plasmid worked. We used electroporation to introduce a reprogramming plasmid and a plasmid carrying the gene for integrase into skin cells and observed colonies of cells that had the characteristics of embryonic stem cells. Because of the great potential of being able to make cells similar to embryonic stem cells out of ordinary adult cells, many scientific groups are working in this field and have made novel contributions. For example, it has been discovered that certain types of adult cells are easier to reprogram than others and that it is possible to substitute small molecules for some of the reprogramming genes. We plan to combine some of these advances with our approach, in order to increase the efficiency of reprogramming, use fewer reprogramming genes, and produce higher quality reprogrammed cells. In the next year, we will carry out that work, as well as thoroughly characterizing the reprogrammed cells that we produce, in terms of their ability to differentiate into different tissue types and show other features of embryonic stem cells. We will also begin experiments to use reprogrammed cells made with these methods to cure genetic diseases.
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.
This project has developed a new method for making stem cells out of ordinary adult cells, such as skin cells. Our project utilizes the discovery that ordinary skin cells can be “reprogrammed” into stem cells that are similar in potential to embryonic stem cells. Such cells can become any type of cell and are useful for understanding and treating many diseases. The reprogramming process is done by introducing four genes that can change the pattern of expression of the genes in a skin cell to that of an embryonic cell.
The reprogramming genes are typically introduced into cells by putting them into viruses that can incorporate, or integrate, themselves into the chromosomes. This process is effective to bring about reprogramming, but it leaves behind viruses embedded in the chromosomes, which can activate genes that cause cancer. To get around this problem, my laboratory has developed a safer method for reprogramming, in which no viruses are used. Instead, we utilize an enzyme that can place a single copy of the reprogramming genes into a safe place in the chromosomes.
In our method, the reprogramming genes are present on small circles of DNA that are easily made and purified from bacteria grown in the laboratory. The circles of DNA, along with DNA that codes for the integration enzyme, are introduced into skin cells. The enzyme causes the reprogramming genes to incorporate into a chromosome at a single, safe location. After the cells are reprogrammed in this way, the reprogramming genes are completely and precisely removed from the chromosomes by introducing another enzyme. The result is reprogrammed cells whose chromosomes are very similar to the chromosomes of the starting cell and contain no added reprogramming genes. These cells appear to be safe to use in the clinic.
In addition, in the project, we developed a method to add a therapeutic gene to the reprogrammed cells. The therapeutic gene is a correct copy of the gene that is mutated in patients with a genetic disease. By adding a correct copy of the mutated gene, the stem cells now make a functional gene product and are healthy. The corrected stem cells can be used for transplantation into the patient to generate healthy tissues.
We demonstrated that cells reprogrammed by our method can be grown in such a way that they form muscle precursor cells that have the capacity to become healthy muscle fibers. We added a therapeutic gene to the reprogrammed cells that provides a correct copy of the gene that is mutated in the limb girdle form of muscular dystrophy. These corrected stem cells will be used in a strategy to repair the muscle damage present in these muscular dystrophy patients